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Methods in
Molecular Biology 1336

Mar Orzáez
Mónica Sancho Medina
Enrique Pérez-Payá Editors

Cyclin-Dependent
Kinase (CDK)
Inhibitors
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Cyclin-Dependent
Kinase (CDK) Inhibitors

Methods and Protocols

Edited by

Mar Orzáez
Medicinal Chemistry, Centro de Investigación Príncipe Felipe, Valencia, Spain

Mónica Sancho Medina

Medicinal Chemistry, Centro de Investigación Príncipe Felipe, Valencia, Spain

Enrique Pérez-Payá
IBV-CSIC and Centro de Investigacion Príncipe Felipe, Valencia, Spain
Editors
Mar Orzáez Mónica Sancho Medina
Medicinal Chemistry Medicinal Chemistry, Centro de Investigación
Centro de Investigación Príncipe Felipe Príncipe Felipe
Valencia, Spain Valencia, Spain

Enrique Pérez-Payá
IBV-CSIC and Centro de Investigacion
Príncipe Felipe
Valencia, Spain

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-2925-2 ISBN 978-1-4939-2926-9 (eBook)
DOI 10.1007/978-1-4939-2926-9

Library of Congress Control Number: 2015944984

Springer New York Heidelberg Dordrecht London

© Springer Science+Business Media, LLC 2016
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
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The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed
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Preface

Proliferation of eukaryotic cells is under control of a series of concerted molecular mecha-

nisms defined as the cell division cycle whose progression is tightly governed by members
of the cyclin-dependent kinase family (CDKs). At different phases of the cell cycle, the
CDKs associate with specific cyclins. These particular CDK/cyclin pairs preferentially phos-
phorylate sets of largely distinct substrates triggering the progression through the cell cycle
phases. For example, CDK2/cyclin E mediated phosphorylation of the tumor suppressor
protein pRb is required for inactivation of the protein and for the subsequent release of E2F
transcription factor, altering the status of E2F-regulated genes from fully repressed to
induced and leading progression through G1 to S phases.
Deregulation of CDK function is a hallmark of cancer. Aberrations in CDK activity
have also been observed in viral infections, neurological diseases, ischemia, and some pro-
liferative disorders. This has led to the intensive research on CDK inhibitors for therapeutic
applications.
Several strategies have been used to inhibit these complexes such as development of
ATP-competitive inhibitors, molecules targeting the CDK/cyclin interaction surfaces or
inhibitors mimicking natural CDK inhibitor proteins (CKIs). However, clinical develop-
ment has not been as successful as expected. More precise details of how inhibitors behave
in different disease models remain to be elucidated and more specific inhibitors with
improved pharmacological characteristics still need to be developed.
Protocols to develop screening assays or to identify novel CDK inhibitors have been
compiled in the first part of the book. Future work in this area will reveal novel targets for
pharmacological interference and could lead to more specific urgently needed cell cycle
inhibitors. The second part of the book describes elaborate procedures to evaluate activity
and mechanism of action of new and/or already identified CDK inhibitors. A number of
expert laboratories have developed specific techniques to study many aspects of CDK inhib-
itors activity in cell culture and in vivo models. The book also compiles protocols to evalu-
ate metabolomics changes associated with inhibitor treatment. Moreover, drug delivery
strategies focused on nanoparticle development to provide alternative internalization sys-
tems for increasing inhibitor efficacy have been also described. We hope this collection will
be a relevant source of information for drug discovery in cell cycle research.
This project would not have been possible without the help of some key people. Thank
you to Dr. John Walker and Anna Rakovsky for providing support along the way. We would
like to thank all the authors for their excellent contributions throughout this project. These
protocols and the notes shared by each author will form a worthwhile tool for scientists
interested in this research field. Finally, we would like to dedicate the book to the memory
of Professor Enrique Pérez-Payá.

Valencia, Spain Mar Orzáez

Mónica Sancho Medina

v
Contents

1 Immunoprecipitation of Cdk–Cyclin Complexes for Determination

of Kinase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Edurne Gallastegui and Oriol Bachs
2 Expression and Purification of Recombinant Cyclins
and CDKs for Activity Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Edurne Gallastegui and Oriol Bachs
3 Expression and Purification of Recombinant CDKs: CDK7, CDK8,
and CDK9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Reena Pinhero and Krassimir Yankulov
4 Preparation of CDK/Cyclin Inhibitor Complexes
for Structural Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Asterios I. Grigoroudis and George Kontopidis
5 Fragment-Based De Novo Design of Cyclin-Dependent
Kinase 2 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Sunil Kumar Tripathi, Poonam Singh, and Sanjeev Kumar Singh
6 Protein-Protein Interaction for the De Novo Design
of Cyclin-Dependent Kinase Peptide Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 59
Karthiga Arumugasamy, Sunil Kumar Tripathi, Poonam Singh,
and Sanjeev Kumar Singh
7 Identification of Cyclin A Binders with a Fluorescent Peptide Sensor . . . . . . . . 67
Elena Pazos, José L. Mascareñas, and M. Eugenio Vázquez
8 Cell Synchronization Techniques to Study the Action of CDK Inhibitors . . . . 85
Beatriz Pérez-Benavente and Rosa Farràs
9 Analysis of CDK Inhibitor Action on Mitochondria-Mediated Apoptosis . . . . . 95
Anna Gortat
10 Evaluating the Effects of CDK Inhibitors in Ischemia–Reperfusion
Injury Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Tatiana Guevara
11 Assessing Cell Cycle Independent Function of the CDK Inhibitor
p21CDKN1A in DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Ilaria Dutto, Micol Tillhon, and Ennio Prosperi
12 Drug Delivery Strategies of Chemical CDK Inhibitors. . . . . . . . . . . . . . . . . . . 141
Daniel Alvira and Laura Mondragón
13 Animal Models for Studying the In Vivo Functions of Cell Cycle CDKs . . . . . 155
Sanjiv Risal, Deepak Adhikari, and Kui Liu
14 Evaluating Chemical CDK Inhibitors as Cell Death Inducers. . . . . . . . . . . . . . 167
Hiroshi Hirai and Yoko Nakatsuru

vii
viii Contents

15 Models for the Study of the Cross Talk Between Inflammation

and Cell Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Laura J. Hoodless, Calum T. Robb, Jennifer M. Felton,
Carl S. Tucker, and Adriano G. Rossi
16 Metabolomic Applications to the Characterization
of the Mode-of-Action of CDK Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Martina Palomino-Schätzlein and Antonio Pineda-Lucena

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Contributors

DEEPAK ADHIKARI • Department of Chemistry and Molecular Biology,

University of Gothenburg, Gothenburg, Sweden
DANIEL ALVIRA • Centre Mediterranéen de Médecine Moléculaire (C3M), Biology and
pathology of melanocyte cells: from skin pigmentation to melanomas,
Institut National de la Santé et de la Recherche Médicale (INSERM), Nice, France
ORIOL BACHS • Department of Cell Biology, Immunology and Neurosciences, University of
Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer
(IDIBAPS), Barcelona, Spain
ILARIA DUTTO • Genome Stability Group, Department of Biology and Biotechnology,
Istituto di Genetica Molecolare del CNR, University of Pavia, Pavia, Italy
ROSA FARRÀS • Oncogenic Signaling Laboratory, Centro de Investigación Príncipe
Felipe de Valencia, Valencia, Spain
JENNIFER M. FELTON • MRC Centre for Inflammation Research, The Queen’s Medical
Research Institute, The University of Edinburgh, Edinburgh, UK
EDURNE GALLASTEGUI • Department of Cell Biology, Immunology and Neurosciences,
University of Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques
August Pi i Sunyer (IDIBAPS), Barcelona, Spain
ANNA GORTAT • Department of Anatomical Pathology, Pharmacology and Microbiology,
University of Barcelona, Barcelona, Spain
ASTERIOS I. GRIGOROUDIS • Department of Biochemistry, Veterinary School,
University of Thessaly, Karditsa, Greece
TATIANA GUEVARA • Laboratory of Peptide and Protein Chemistry, Centro de Investigación
Príncipe Felipe, Valencia, Spain
HIROSHI HIRAI • Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba,
Ibaraki, Japan
LAURA J. HOODLESS • MRC Centre for Inflammation Research, The Queen’s Medical
Research Institute, The University of Edinburgh, Edinburgh, UK
KARTHIGA ARUMUGASAMY • Computer Aided Drug Designing and Molecular Modeling Lab,
Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu, India
GEORGE KONTOPIDIS • Department of Biochemistry, Veterinary School, University of
Thessaly, Karditsa, Greece
KUI LIU • Department of Chemistry and Molecular Biology, University of Gothenburg,
Gothenburg, Sweden
JOSÉ L. MASCAREÑAS • Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CIQUS), Departamento de Química Orgánica, Universidade de Santiago
de Compostela, Santiago de Compostela, Spain
LAURA MONDRAGÓN • Centre Mediterranéen de Médecine Moléculaire (C3M),
Biology and pathology of melanocyte cells: from skin pigmentation to melanomas,
Institut National de la Santé et de la Recherche Médicale (INSERM), Nice, France

ix
x Contents

YOKO NAKATSURU • Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba,
Ibaraki, Japan
MARTINA PALOMINO-SCHÄTZLEIN • Structural Biochemistry Laboratory, Advanced
Therapies Program, Centro de Investigación Príncipe Felipe, Valencia, Spain
ELENA PAZOS • Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CIQUS), Departamento de Química Orgánica, Universidade de
Santiago de Compostela, Santiago de Compostela, Spain
BEATRIZ PÉREZ-BENAVENTE • Oncogenic Signaling Laboratory, Centro de Investigación
Príncipe Felipe de Valencia, Valencia, Spain; Biochemistry and Molecular Biology
Department, Universidad de Valencia, Valencia, Spain
ENRIQUE PÉREZ-PAYÁ • IBV-CSIC and Centro de Investigación Príncipe Felipe, Valencia,
Spain
ANTONIO PINEDA-LUCENA • Structural Biochemistry Laboratory, Advanced Therapies
Program, Centro de Investigación Príncipe Felipe, Valencia, Spain
REENA PINHERO • Department of Molecular Biology and Genetics, University of Guelph,
Guelph, ON, Canada
ENNIO PROSPERI • Genome Stability Group, Department of Biology and Biotechnology,
Istituto di Genetica Molecolare del CNR, University of Pavia, Pavia, Italy
SANJIV RISAL • Department of Chemistry and Molecular Biology, University of Gothenburg,
Gothenburg, Sweden
CALUM T. ROBB • MRC Centre for Inflammation Research, The Queen’s Medical Research
Institute, The University of Edinburgh, Edinburgh, UK
ADRIANO G. ROSSI • MRC Centre for Inflammation Research, The Queen’s Medical
Research Institute, The University of Edinburgh, Edinburgh, UK
POONAM SINGH • Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow,
India
SANJEEV KUMAR SINGH • Computer Aided Drug Designing and Molecular Modeling Lab,
Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu, India
MICOL TILLHON • Genome Stability Group, Department of Biology and Biotechnology,
Istituto di Genetica Molecolare del CNR, University of Pavia, Pavia, Italy
SUNIL KUMAR TRIPATHI • Computer Aided Drug Designing and Molecular Modeling Lab,
Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu, India
CARL S. TUCKER • BHF Centre for Cardiovascular Science, The Queen’s Medical Research
Institute, The University of Edinburgh, Edinburgh, UK
M. EUGENIO VÁZQUEZ • Centro Singular de Investigación en Química Biolóxica e
Materiais Moleculares (CIQUS), Departamento de Química Orgánica,
Universidade de Santiago de Compostela, Santiago de Compostela, Spain
KRASSIMIR YANKULOV • Department of Molecular Biology and Genetics,
University of Guelph, Guelph, ON, Canada
 Chapter 1

Immunoprecipitation of Cdk–Cyclin Complexes

for Determination of Kinase Activity
Edurne Gallastegui and Oriol Bachs

Abstract
Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription.
The activity of some of them is deregulated in tumor cells and to find specific inhibitors is an important goal
to be achieved. We report here the current methods to determine their in vitro activity in order to facilitate
the identification of specific inhibitors. Mainly, the activity can be determined by using immunoprecipitates
from cell samples with antibodies against specific Cdks as a source of the enzymes.

Key words Cdk2, Cdk4, Cdk6, Cdk1, Cyclin A, Cyclin B, Cyclin D, Kinase assay

1 Introduction

Cyclin dependent kinases (CDKs) are serine/threonine kinases

characterized by needing a regulatory subunit (cyclin) that by asso-
ciating with the enzyme provides domains necessary for the cata-
lytic activity [1]. This family of kinases includes 20 members that
recently have been renamed as Cdk1 through to Cdk20 [2]. From
the functional point of view this family of kinases participates in
two major cellular roles: cell division cycle and transcription. The
analysis of evolutionary expansion of the Cdk family in mammals
allows to classify these enzymes into three cell-cycle related sub-
families: Cdk1, Cdk4, and Cdk5 and five transcriptional subfami-
lies: Cdk7, Cdk8, Cdk9, Cdk11, and Cdk20 [3]. Most of these
Cdks bind one or a few cyclins that confers to these specific com-
plexes a functional specialization [1]. The cyclin family (the regula-
tory subunits of Cdks) contains approximately 29 proteins in
humans clustered in three major groups: cyclin B group, mostly
associated to cell cycle related Cdks, the cyclin I group, the part-
ners of Cdk5 and the cyclin C group, the major partners of tran-
scriptional Cdks. Thus, a significant number of specific Cdk–cyclin
complexes can be generated and it is assumed that each one of

Mar Orzáez et al. (eds.), Cyclin-Dependent Kinase (CDK) Inhibitors: Methods and Protocols, Methods in Molecular Biology,
vol. 1336, DOI 10.1007/978-1-4939-2926-9_1, © Springer Science+Business Media, LLC 2016

1
2 Edurne Gallastegui and Oriol Bachs

these complexes have, at least in some degree, specific substrate

specificity [1]. As a consequence, the different Cdk–cyclin com-
plexes play specific roles in cellular functions. In addition to the
regulation by cyclins, Cdk activity is also regulated by other mech-
anisms that include phosphorylation of specific amino acid resi-
dues, acetylation, and binding to proteins called Cdk-inhibitors
(CKIs) [4, 5]. A relevant phosphorylation that directly affects Cdk
activity is that of the threonine residue 160 in Cdk2 (or its equiva-
lent in other Cdks) by CAK (Cdk activating kinase). This phos-
phorylation stabilizes the activated form of the kinase heterodimer.
However, this is not general and for instance Cdk5 does not require
this phosphorylation for full activity [6]. A second relevant regula-
tory phosphorylation is that occurring at specific residues Thr14
and Tyr15 in Cdk2 (or their equivalents in the other Cdks). These
phosphorylations are performed by Wee1 and Myt1 kinases and
inhibit the activity of several family members [7]. Another regula-
tory mechanism of Cdk activity is acetylation. It has been reported
that at least Cdk2 can be acetylated by the acetyltransferase PCAF
at a specific Lys residue of the catalytic center and the kinase activ-
ity is inhibited because this Lys residue is critical for the interaction
with ATP [5]. This acetylation has not been demonstrated to occur
in all Cdks. However, because it is a conserved residue it can be
hypothesized that acetylation of this residue can be a general
mechanism of Cdk inhibition [8].
Finally, the cell cycle related CDKs can also be negatively regu-
lated by binding to low molecular weight proteins of the INK4 or
Cip/Kip families of inhibitors [9]. INK4 proteins (p16INK4a,
p15INK4b, p18INK4c, and p19INK4d) are specific for the Cdk4 subfam-
ily and interact with the monomeric Cdks preventing the activation
by the D-type cyclins or by CAK. Members of the Cip/Kip family
of inhibitors (p21Cip1, p27Kip1, and p57Kip2) associate with both the
Cdk and the cyclin subunits and inhibit the kinase activity by intro-
ducing a domain into the catalytic center of the Cdks, thus block-
ing ATP association [9]. It has been shown that p27 and p21 can
be phosphorylated at specific Tyr residues by members of the Src
family of protein kinases. When phosphorylated at these Tyr resi-
dues, p27 and p21 associated with Cdk–cyclin complexes cannot
fully inhibit Cdk activity. Thus, under these conditions the com-
plexes remain partially activated [10, 11]. All these aspects of the
regulation of Cdk activity have to be taken into account when its
activity has to be determined.
In this chapter we describe a general protocol to determine
in vitro Cdk activity by using Cdk–cyclin complexes obtained by
immunoprecipitation (IP) of cell samples with antibodies against
specific Cdks or cyclins as a source of the enzymes. When the activ-
ity has to be analyzed after IP of Cdk–cyclin complexes the first
aspect to be considered is the antibody that has to be used for the
IP. When a Cdk can associate with several cyclins, the use of the
 Determination of Kinase Activity 3

antibody against the Cdk will allow determining the activity of all
complexes containing this Cdk. On the contrary, to only analyze
the activity of a specific Cdk–cyclin complex, the antibody against
the specific cyclin of the complex has to be used. However, in some
cases a specific cyclin can bind to several Cdks. For instance, cyclin
A can associate with both Cdk2 and Cdk1. Thus, in these cases it
is better to use antibodies against the Cdk or to perform several
determinations using antibodies against the Cdk and additionally
also antibodies against each cyclin and then with all results the
interested activity can be calculated. Another consideration is that
on depending of the status of the cell cultures used for the IPs a
percentage of Cdk–cyclin complexes can be associated with CKIs.
Additionally, these associated CKIs (more specifically p27 and
p21) can be Tyr phosphorylated. Thus, immunoprecipitates may
contain a heterogeneous population of free Cdk or free cyclin (on
depending of the antibody used), free Cdk–cyclin complexes and
complexes associated with CKIs that can be Tyr phosphorylated or
not. Finally, Cdks in the immunoprecipitates can be acetylated or
phosphorylated at T160 or/and T14/Tyr15.
We describe here the protocols for the in vitro determination
of the activities of relevant Cdk–cyclin complexes involved in cell
cycle regulation as Cdk1, Cdk2, and Cdk4/6. These activities will
be determined by using complexes obtained by IP of cellular
extracts by using antibodies against Cdks.

2 Materials

2.1 Immunopreci- 1. HAT buffer + Inhibitors: 50 mM Tris–HCl pH 8, 50 mM KCl,

pitation and Kinase 0.1 mM EDTA pH 8, 5 % glycerol, 0.5 % NP-40, 0.5 μg/ml
Assay for aprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 0.1 mM Na3VO4.
Determination of Cdk2 2. 10× Kinase buffer Cdk2: 250 mM HEPES pH 7.4, 100 mM
Activity MgCl2.
3. 1× Kinase buffer Cdk2: 25 mM HEPES pH 7.4, 10 mM
MgCl2.
4. Kinase assay reaction buffer: 2 μg Histone H1 (Cdk2 sub-
strate), 12.5 μM ATP, kinase buffer 10×, 1 μCi [ɣ-32P], 2 mM
DTT, Milli-Q water.
5. 4× Loading buffer: 40 mM NaPi pH 7.0, 40 % glycerol, 10 %
SDS, 2 mg/ml DTT, 0.67 mg/ml bromophenol blue.
6. Coomassie Blue staining: 2.5 mg/ml Coomassie Brilliant Blue,
45 % methanol, 10 % acetic acid.
7. Destaining solution: 50 % methanol, 10 % acetic acid.
8. Bradford reagent.
9. Protein A/G Agarose Beads.
4 Edurne Gallastegui and Oriol Bachs

10. Antibodies: Cdk2 (Santa Cruz, sc-163), Cyclin A (Santa Cruz,

sc-596), Cyclin E (Santa Cruz, sc-481).
11. X-ray Films.
12. Sonicator (Bioruptor).
13. Microcentrifuge.

2.2 Immunopreci- 1. Lysis buffer D4 + Inhibitors: 50 mM HEPES pH 7.5, 150 mM

pitation and Kinase NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.1 % Tween 20, 10 %
Assay for glycerol, 1 mM DTT, 10 mM β-glycerol phosphate, 0.1 mM
Determination Na3VO4, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM
of Cdk4/6 Activity PMSF.
2. 10× Kinase buffer Cdk4/6: 500 mM HEPES pH 7.4, 100 mM
MgCl2, 25 mM EGTA, 10 mM DTT.
3. 1× Kinase buffer Cdk4/6: 50 mM HEPES pH 7.4, 10 mM
MgCl2, 2.5 mM EGTA, 1 mM DTT.
4. Kinase assay reaction buffer: 3 μg GST-pRB (aa. 379–928)
(Cdk4/6 substrate), 15 μM ATP, kinase buffer 10×, 2 μCi
[ɣ-32P], 1 mM DTT, Milli-Q water.
5. 4× Loading buffer: 40 mM NaPi pH 7.0, 40 % glycerol, 10 %
SDS, 2 mg/ml DTT, 0.67 mg/ml bromophenol blue.
6. Coomassie staining: 2.5 mg/ml Coomassie Brilliant Blue, 45 %
Methanol, 10 % Acetic acid.
7. Destaining solution: 50 % Methanol, 10 % Acetic acid.
8. Bradford reagent.
9. Protein A/G Agarose Beads.
10. Antibodies: Cdk4 (Santa Cruz, sc-260), Cdk6 (Santa Cruz,
sc-7181).
11. X-ray Films.
12. Sonicator (Bioruptor).
13. Microcentrifuge.

2.3 Immunopreci- 1. HAT buffer + Inhibitors: 50 mM Tris–HCl pH 8, 50 mM KCl,

pitation and Kinase 0.1 mM EDTA pH 8, 5 % glycerol, 0.5 % NP-40, 0.5 μg/ml
Assay for aprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 0.1 mM Na3VO4.
Determination of Cdk1 2. 10× Kinase buffer Cdk1: 200 mM HEPES pH 7.4, 100 mM
Activity magnesium acetate.
3. 1× Kinase buffer Cdk2: 20 mM HEPES pH 7.4, 10 mM mag-
nesium acetate.
4. Kinase assay reaction buffer: 2 μg Histone H1 (Cdk2 sub-
strate), 12.5 μM ATP, kinase buffer 10×, 1 μCi [ɣ-32P], 2 mM
DTT, Milli-Q water.
5. 4× Loading buffer: 40 mM NaPi pH 7.0, 40 % glycerol, 10 %
SDS, 2 mg/ml DTT, 0.67 mg/ml bromophenol blue.
 Determination of Kinase Activity 5

6. Coomassie Blue staining: 2.5 mg/ml Coomassie Brilliant Blue,

45 % methanol, 10 % acetic acid.
7. Destaining solution: 50 % methanol, 10 % acetic acid.
8. Bradford reagent.
9. Protein A/G Agarose Beads.
10. Antibodies: Cdk1 (Abcam, ab32384).
11. X-ray Films.
12. Sonicator (Bioruptor).
13. Microcentrifuge.

3 Methods

3.1 Immunopreci- 1. Cells are lysed using HAT buffer + inhibitors for 30 min on ice
pitation and Kinase (see Note 1).
Assay for 2. After incubation, centrifuge the samples and clarify at
Determination of Cdk2 11,700 × g for 10 min at 4 °C.
Activity 3. Transfer the supernatants containing whole cell protein extract
3.1.1 Cell Lysis to new tubes.
4. Protein concentration of each sample is quantified using the
Bradford assay.

3.1.2 Immunoprecip- 1. Immunoprecipitate 500 μg of whole cell proteins with 2 μg of

itation Cdk2, Cyclin A, or Cyclin E antibody for 16 h. IgG antibody
(2 μg) is used as a negative control. The final volume of each
IP is 500 μl in HAT buffer + inhibitors.
2. After antibody incubation, add 30 μl of protein A-Sepharose
or protein G-Sepharose (see Note 2) to the samples that are
subsequently incubated for 1 h at 4 °C on rotation.
3. Wash the beads three times with HAT buffer. At this step, each
sample (including the beads) is split in two tubes; one of them
is used to perform the kinase assay and the other one to per-
form a western blot to detect the amount of immunoprecipi-
tated Cdk2.
4. Wash the tubes used for the kinase assay once with 1× kinase
buffer.

3.1.3 Kinase Assay 1. Add 30 μl of kinase assay reaction buffer, prepared as explained
above (see Subheading 2), to each tube (see Note 3).
2. Perform the kinase assay at 30 °C for 30 min and then stop the
reaction by adding 10 μl of 4× loading buffer.
3. Centrifuge the samples for 30 s at 2000 × g to remove the
beads.
6 Edurne Gallastegui and Oriol Bachs

3.1.4 Polyacrylamide Gel 1. Run the samples in a 12 % polyacrylamide gel at 120 V for 1 h
Electrophoresis and 15 min.
and Coomassie Blue 2. Stain the gel with Coomassie Blue staining solution for 15 min
Staining in a glass tray and then remove this solution and replace with
Coomassie destaining solution for 16 h (overnight) in a shaker
machine (see Note 4).
3. Place on the top of the gel a maximum resolution film recom-
mended for 32P detection (Kodak Biomax) and expose between
2 and 24 h (see Note 5) at −80 °C to detect 32P-H1.
4. After exposing the film, develop it in a developer machine.
The amount of immunoprecipitated Cdk2 protein in the west-
ern blot and the amount of H1 protein (Cdk2 substrate) observed
in the Coomassie stained gel are used as a loading control for the
kinase assay.

3.2 Immunopreci- 1. Cells are lysed using lysis buffer D4 + inhibitors for 30 min on
pitation and Kinase ice (see Note 6).
Assay for 2. Sonicate the samples twice for 10 s at 4 °C.
Determination
3. After sonication, centrifuge the samples and clarify at
of Cdk4/6 Activity
11,700 × g for 10 min at 4 °C.
3.2.1 Cell Lysis 4. Transfer the supernatants containing whole cell protein extract
to new tubes.
5. Protein concentration of each sample is quantified using the
Bradford assay.

3.2.2 Immunopreci- 1. Immunoprecipitate 500 μg of whole cell proteins with 2 μg of

pitation Cdk4 or Cdk6 antibody for 16 h. IgG antibody (2 μg) is used
as a negative control. The final volume of each IP is 500 μl in
lysis buffer D4 + inhibitors.
2. After antibody incubation, add 30 μl of protein A-Sepharose
or protein G-Sepharose (see Note 2) to the samples that are
subsequently incubated for 1 h at 4 °C on rotation.
3. Wash the beads three times with lysis buffer D4. At this step,
each sample (including the beads) is split in two tubes; one of
them is used to perform the kinase assay and the other one to
perform a western blot to detect the amount of immunopre-
cipitated Cdk4/6.
4. Wash the tubes used for the kinase assay once with 1× kinase
buffer Cdk4/6.

3.2.3 Kinase Assay 1. Add 30 μl of kinase assay reaction buffer, prepared as explained
above (see Subheading 2), to each tube (see Note 3).
2. Perform the kinase assay at 30 °C for 30 min and then stop the
reaction by adding 10 μl of 4× loading buffer.
3. Centrifuge the samples for 30 s at 3000 rpm to remove the
beads.
 Determination of Kinase Activity 7

3.2.4 Polyacrylamide 1. Run the samples in a 10 % polyacrylamide gel at 120 V for 1 h

Gel Electrophoresis and 15 min.
and Coomassie Staining 2. Stain the gel with Coomassie Blue staining solution for 15 min
in a glass tray and then remove this solution and replace with
Coomassie destaining solution for 16 h (overnight) in a shaker
machine (see Note 4).
3. Place on the top a maximum resolution film recommended for
32
P detection (Kodak Biomax) and expose between 2 and 24 h
(see Note 5) at −80 °C to detect 32P-GST-pRB (379–928).
4. After exposing the film, develop it in a developer machine.
The amount of immunoprecipitated Cdk4/6 proteins in the
western blot and the amount of GST-pRB (aa. 379–928) protein
(Cdk4/6 substrate) observed in the Coomassie stained gel are
used as a loading control for the kinase assay.

3.3 Immunopreci- Kinase assay of Cdk1 is performed like Cdk2 kinase assay with an
pitation and Kinase exception: 500 μg of whole cell proteins is immunoprecipitated
Assay for with 1 μg of Cdk1 or Cyclin B1 antibody for 16 h and 1 μg of IgG
Determination antibody is used as a negative control.
of Cdk4/6 Activity

4 Notes

1. We use 200 μl of HAT buffer to lyse one million cells. For

each IP, 500 μg of protein is used. Thus, the number of start-
ing cells depends on the cell type. The HAT buffer can be
prepared and stored at 4 °C, but the inhibitors must be added
at the beginning of the protocol (0.5 μg/ml aprotinin, 10 μg/
ml leupeptin, 1 mM PMSF, 0.1 mM Na3VO4).
2. Protein A-sepharose or protein G-sepharose would be used
depending on the species of the antibody we use (Table 1).

Table 1
Protein A/G binding affinities

Species Protein A Protein G

Human ++++ ++++
Mouse ++ −
Rat − ++
Rabbit ++++ +++
Goat − ++
Sheep +/− ++
8 Edurne Gallastegui and Oriol Bachs

3. Half-life of 32P is around 15 days. This means that every

2 weeks, the amount of radioactivity that it is needed for kinase
assays, has to be doubled. This has to be taken into account
when the kinase assay reaction buffer is prepared.
4. Coomassie Blue staining and destaining solutions can be
reused several times before been wasted.
5. Time of exposure will depend on the amount of Cdk2 or
Cdk4/6 and their activity in the cells we are studying. We
recommend to do a short exposure and according to the
result, next step would be to increase this time.
6. We use 200 μl of lysis buffer D4 buffer to lyse one million
cells. For each IP, 500 μg of protein is used. Thus, the number
of starting cells depends on the cell type. The lysis buffer can
be prepared and stored at 4 °C but DTT and the inhibitors
must be added at the beginning of the protocol (10 mM
β-glycerol phosphate, 0.1 mM Na3VO4, 1 μg/ml aprotinin,
10 μg/ml leupeptin, 1 mM PMSF).

References
1. Malumbres M (2014) Cyclin-dependent 7. Malumbres M, Barbacid M (2005) Mammalian
kinases. Genome Biol 15:122–131 cyclin-dependent kinases. Trends Biochem Sci
2. Malumbres M, Harlow E, Hunt T, Hunter T, 30:630–641
Lahti JM, Manning G, Morgan DO, Tsai L-H, 8. Russo AA, Jeffrey PD, Patten AK, Massagué J,
Wolgemuth DJ (2009) Cyclin-dependent Pavletich NP (1996) Crystal structure of the
kinases: a family portrait. Nat Cell Biol p27Kip1 cyclin-dependent-kinase inhibitor
11:1275–1276 bound to the cyclin A-Cdk2 complex. Nature
3. Cao L, Chen F, Yang X, Xu W, Xie J, Yu L 382:325–331
(2014) Phylogenetic analysis of CDK and 9. Pavletich NP (1999) Mechanisms of cyclin-
cyclin proteins in premetazoan lineages. BMC dependent kinase regulation: structures of
Evol Biol 14:10 Cdks, their cyclin activators, and Cip and
4. Morgan DO (1997) Cyclin-dependent kinases: INK4 inhibitors. J Mol Biol 287:821–828
engines, clocks, and microprocessors. Annu 10. Grimmler M, Wang Y, Mund T, Cilensek Z,
Rev Cell Dev Biol 13:261–291 Keidel E-M, Waddell MB, Jäkel H, Kullmann
5. Mateo F, Vidal-Laliena M, Canela N, Zecchin M, Kriwacki RW, Hengst L (2007) Cdk-
A, Martínez-Balbás M, Agell N, Giacca M, inhibitory activity and stability of p27Kip1 are
Pujol MJ, Bachs O (2009) The transcriptional directly regulated by oncogenic tyrosine
co-activator PCAF regulates Cdk2 activity. kinases. Cell 128:269–280
Nucleic Acids Res 37:7072–7084 11. Chu I, Sun J, Arnaout A, Kahn H, Hanna W,
6. Echalier A, Endicott JA, Noble MEM (2010) Narod S, Sun P, Tan C-K, Hengst L,
Recent developments in cyclin-dependent Slingerland J (2007) p27 phosphorylation by
kinase biochemical and structural studies. Src regulates inhibition of cyclin E-Cdk2. Cell
Biochim Biophys Acta 1804:511–519 128:281–294
 Chapter 2

Expression and Purification of Recombinant Cyclins

and CDKs for Activity Evaluation
Edurne Gallastegui and Oriol Bachs

Abstract
Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription.
Their activity is mainly regulated by association with regulatory subunits named cyclins but their activities
are also regulated by phosphorylation, acetylation, and the association with specific inhibitory proteins
(CKIs). The activity of different Cdks is deregulated in many different type of tumors, and thus, Cdks are
considered targets for antitumoral therapy. For large screenings of inhibitors the use of purified recombi-
nant Cdks and cyclins is recommended. We report here the current methods to determine their in vitro
activity for large screenings of inhibitors.

Key words Cdk1, Cdk2, Cyclin A, Cyclin B, Kinase assay

1 Introduction

Regarding cell cycle regulation, the most important cyclins are the
D-type cyclins that include three members (cyclins D1, D2, and
D3), cyclin E, cyclin A, and cyclin B [1]. During cell cycle prog-
ression different Cdk–cyclin complexes are generated each one
operating at specific moments along the cell cycle [2]. Specifically,
Cdk4/6–cyclins D complexes are activated during the G1 phase of
the cell cycle and they are responsible of phosphorylating members
of the pocked proteins family (pRb, p107, and p130) that are in
transcriptional repressor complexes [3]. Subsequent phosphoryla-
tion of these complexes, by Cdk2–cyclin E, disrupts them inducing
the transcription of genes encoding proteins necessary for DNA
replication and mitosis [4]. Cdk2–cyclin E complexes are also
involved in the triggering of DNA replication. Cdk2–cyclin A are
subsequently activated and are necessary for the progression of
DNA replication during the S phase of the cell cycle. Finally, Cdk1–
cyclin A and Cdk1–cyclin B participate in the triggering and
progression of mitosis [5].

Mar Orzáez et al. (eds.), Cyclin-Dependent Kinase (CDK) Inhibitors: Methods and Protocols, Methods in Molecular Biology,
vol. 1336, DOI 10.1007/978-1-4939-2926-9_2, © Springer Science+Business Media, LLC 2016

9
10 Edurne Gallastegui and Oriol Bachs

The activities of different Cdks are deregulated in many different

types of tumors, so they are considered relevant targets for antitu-
moral therapy [6]. To identify specific Cdk inhibitors, protocols to
determine their activity have been developed. The in vitro Cdk
activity is mainly determined by two general protocols. The first is
by using Cdk–cyclin complexes obtained by immunoprecipitation
(IP) of cell samples with antibodies against specific Cdks or cyclins
as a source of the enzymes (Chapter 1). The second is by using
purified recombinant Cdks and cyclins. When performing kinase
assays using purified recombinant proteins, a more defined com-
position of the complexes can be achieved. When proteins are
expressed in and purified from bacteria, the post-translational
modifications (phosphorylation and acetylation) of Cdks are not
produced and additionally no CKIs are associated. However,
because the activating phosphorylation at T160 is not produced,
the activity of the complexes that need this phosphorylation for full
activity only would display a reduced activity. This problem can be
solved by in vitro phosphorylating this T160 residue by incubation
of the purified Cdk with the CAK enzyme previously to mix the
Cdk with the selected cyclin. Finally, in vitro association of specific
Cdks with their cyclin partners in some cases might be highly inef-
ficient. For instance, the association of Cdk4 with D-type cyclins is
one of these cases. The CKIs p21 and p27 can work as adaptor
proteins that stimulate the interaction between both subunits [7].
However, because p21 and p27 also work as inhibitors of these
complexes the in vitro activity of these complexes using purified
recombinant proteins is difficult.
We describe here the protocols to determine Cdk1 and Cdk2
activities by using recombinant proteins expressed in and purified
from bacteria. These protocols are recommended when the aim is
to screen high amount of products on trying to identify specific
inhibitors of these complexes. To analyze the activity of other
Cdk–cyclin complexes, specific modifications of the protocols have
to be done.

2 Materials

2.1 Expression 1. LB (Luria–Bertani) medium: 1 % tryptone, 0.5 % yeast extract,

and Purification 0.5 % NaCl (Autoclave before use).
of Recombinant 2. Antibiotics: ampicillin (final concentration: 50 μg/ml) and
Cyclins and Cdks chloramphenicol (final concentration: 20 μg/ml).
for Determination 3. Bacteria strain: BL21 (DE3).
of Kinase Activity
4. IPTG.
5. Lysis buffer NETN + Inhibitors: 20 mM Tris–HCl pH 8,
100 mM NaCl, 1 mM EDTA, 0.5 % NP-40, 0.5 μg/ml apro-
tinin, 10 μg/ml leupeptin, 1 mM PMSF, 0.1 mM Na3VO4.
 Purification of Cyclin A, Cyclin B, Cdk1 and Cdk2 11

6. Glutathione-Sepharose-4B resin.
7. Elution buffer: 50 mM Tris–HCl pH 9.6, 120 mM NaCl,
20 mM glutathione reduced.
8. 1× PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
2 mM KH2PO4.
9. Bradford reagent.

3 Methods

3.1 Protein Cdk and cyclin cDNA must be cloned in a pGEX plasmid. This
Expression kind of plasmids is used to express high amounts of proteins fused
to glutathione S-transferase (GST).
1. Transform pGEX plasmids containing cDNAs of interest in
BL21 (DE3) and grow in LB medium with ampicillin and
chloramphenicol at 37 °C with shaking to mid-log phase
(OD600nm 0.8).
2. Induce the expression by the addition of isopropyl 1-thio-d-
galactopyranoside (IPTG) at a final concentration of 0.5 mM
and incubate the culture for 4 h with shaking at room
temperature.
3. To test the induction, run 30 μl of non-induced and induced
bacteria culture in a polyacrylamide gel and stain with Coomassie
Blue.

3.2 Protein 1. Harvest the bacteria by centrifugation at 4000 × g at 4 °C for

Purification 10 min.
2. Suspend the pellet in NENT buffer + Inhibitors (see Note 1).
3. Froze the bacterial cells at −80 °C and thaw on ice three times.
4. Lyse the cells by sonication four times for 10 s.
5. Perform a centrifugation at 24,000 × g at 4 °C for 15 min.
6. Do the purification using affinity chromatography by incuba-
ting the supernatant with Glutathione-Sepharose-4B resin in a
column. For every 5 mg of protein 1 ml of Glutathione-
Sepharose-4B has to be used (see Note 2).
7. Incubate the mix with shaking at 4 °C for 1 h.
8. Wash three times with NENT (final volume of 100 ml).
9. At this point, GST from recombinant proteins can be removed
if needed but to perform the kinase assay this step is not neces-
sary (see Note 3).
10. Add 5 ml of elution buffer and incubate with shaking at 4 °C
for 10 min.
12 Edurne Gallastegui and Oriol Bachs

11. Protein is obtained after centrifuging at 2000 × g at 4 °C for

5 min and keeping the supernatant.
12. Dialyze the purified recombinant proteins against PBS and cal-
culate the concentration by Bradford assay.

3.3 Kinase Assay 1. Mix 400 nM of Cdk protein and the same concentration
of cyclin protein in a tube and incubate for 10 min on ice
(as many tubes as reactions we want to perform are prepared at
this step). If the effect of some inhibitor wants to be studied, it
has to be added to the mix at this point.
2. Kinase assay and polyacrylamide gel electrophoresis are per-
formed as previously described (see Chapter 1), depending on
the Cdk–cyclin activity to be determined. For Cdk2, Cdk4/6,
and Cdk1 activities see Subheadings 3.1, 3.2, and 3.3, respec-
tively. For material related to Cdk2, Cdk4/6, and Cdk1 kinase
assays see Subheadings 2.1, and Subheadings 2.2, and 2.3,
from Chapter 1 (E. Gallastegui and O. Bachs).

4 Notes

1. The NETN buffer can be prepared and stored at 4 °C, but the
inhibitors must be added at the beginning of the protocol
(0.5 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF,
0.1 mM Na3VO4).
2. 5 mg of protein is equal to a starting volume of bacterial culture
of 0.5–1 l, approximately.
3. If desired, GST can be removed from the GST fusion proteins
by digestion with thrombin protease.

References

1. Malumbres M, Barbacid M (2005) Mammalian and redundancy in the mitotic entry network.
cyclin-dependent kinases. Trends Biochem Sci J Cell Biol 185:193–202
30:630–641 6. Asghar U, Witkiewicz AK, Turner NC,
2. Morgan DO (1997) Cyclin-dependent kinases: Knudsen ES (2015) The history and future
engines, clocks, and microprocessors. Annu Rev of targeting cyclin-dependent kinases in can-
Cell Dev Biol 13:261–291 cer therapy. Nat Rev Drug Discov 14:
3. Malumbres M (2014) Cyclin-dependent kinases. 130–146
Genome Biol 15:122–131 7. LaBaer J, Garrett MD, Stevenson LF,
4. Rubin SM (2013) Deciphering the retino- Slingerland JM, Sandhu C, Chou HS, Fattaey
blastoma protein phosphorylation code. Trends A, Harlow E (1997) New functional activities
Biochem Sci 38:12–19 for the p21 family of CDK inhibitors. Genes
5. Lindqvist A, Rodríguez-Bravo V, Medema RH Dev 11:847–862
(2009) The decision to enter mitosis: feedback
 Chapter 3

Expression and Purification of Recombinant CDKs:

CDK7, CDK8, and CDK9
Reena Pinhero and Krassimir Yankulov

Abstract
Cyclin-dependent kinases have established roles in the regulation of cell cycle, in gene expression and in
cell differentiation. Many of these kinases have been considered as drug targets and numerous efforts have
been made to develop specific and potent inhibitors against them. The first step in all of these attempts and
in many other biochemical analyses is the production of highly purified and reliable kinase, most frequently
in a recombinant form. In this chapter we describe our experience in the cloning, expression, and purifica-
tion of CDKs using CDK7/CycH, CDK8/CycC, and CDK9/CycT1 as an example.

Key words Cyclin-dependent kinases, CDK expression, CDK purification

1 Introduction

Cyclin-dependent kinases (CDKs) are Ser/Thr protein kinases,

which are critically dependent on their association with a cyclin part-
ner [1–3]. CDKs have a firmly established role as key regulators of
the division of cells. Their cyclin partners are expressed and degraded
in tightly regulated temporal fashion thus furnishing the orches-
trated oscillating kinase activities required for the proper progression
though the cell cycle. Other members of the CDK family play roles
in gene transcription and in cell differentiation [3]. Some of the
cyclins that couple with these kinases do not display oscillating abun-
dance, but still remain critical for the activity of the kinase. Most
cyclins associate with one or two kinases [3]; however, some of the
budding yeast CDKs can associate with as many as nine cyclins [2].
In addition to association with cyclins, CDKs are regulated by
a variety of mechanisms including phosphorylation, association
with inhibitory proteins and assembly factors, and protein degra-
dation [1]. In particular, the activity of many CDKs is dependent
on their phosphorylation by CAK (CDK Activating Kinase). CAK
itself is a CDK, CDK7/CycH [4]. The phosphorylation by CAK
and other posttranslational modifications provide yet another layer

Mar Orzáez et al. (eds.), Cyclin-Dependent Kinase (CDK) Inhibitors: Methods and Protocols, Methods in Molecular Biology,
vol. 1336, DOI 10.1007/978-1-4939-2926-9_3, © Springer Science+Business Media, LLC 2016

13
14 Reena Pinhero and Krassimir Yankulov

of complexity in the regulation of CDKs and pose challenges to the

expression of active recombinant kinases that can be used in vitro.
Many studies on CDKs have been carried out with natural or
epitope-tagged kinases that have been immunoprecipitated from
cell extracts. This approach benefits from the natural environment
for posttranslational modifications of the enzymes. Normally, the
presence of contaminating kinase in such preps is addressed by
testing for kinase activity in a blank control. Additional reassurance
is provided through the use of specific kinase inhibitors or the use
of multiple substrates. However, many CDKs exist in vivo in more
than one complex (see below) and some of these complexes display
substantial variations in their substrate preferences [5–9]. The
uncertainty on the homogeneity of these kinase preparations often
obfuscates the correct interpretation of the data. Clearly, these
small-scale procedures have provided valuable insights, but are
incompatible with large-scale analyses including the search and
characterization of inhibitors and drug candidates.
Here we describe strategies for large-scale expression and puri-
fication of recombinant CDKs with a focus on CDK7/CycH,
CDK8/CycC, and CDK9/CycT1 (see Fig. 1 for outline). These
kinases do not directly regulate the progression through the cell
cycle. CDK7 exits in several forms including a core tripartite
CDK7/CycH/MAT1 complex known as CAK (CDK activating
kinase) and as a component in the general pol II transcription fac-
tor TFIIH [10, 11]. CDK8/CycC exists as a bipartite complex,
but has also been found in a variety of complexes that contain
RNApol II and general RNApol II transcription factors [12].
CDK9 has been initially identified as P-TEFb (Positive Transcription
Elongation Factor-b) [13, 14]. Independently, CDK9/CycT1 has
been isolated as the HIV-tat associated kinase TAK [15, 16]. All
these kinases have multiple targets, but share the carboxy-terminal
domain (CTD) of RNA pol II as a common substrate [10].
As mentioned earlier, most CDKs are regulated by posttransla-
tional modifications. The enzymes that exert these modifications
are not available in prokaryotic cells. Consequently, the expression
and/or assembly of CDKs in E. coli frequently generates inactive
kinases. Practice has shown that insect cells, albeit more expensive
and cumbersome, provide the proper environment for the expres-
sion of active CDKs. We recommend insect Sf9 cells and baculovi-
rus vectors for the expression of these enzymes.

2 Materials

2.1 Growth 1. 1× Grace’s Medium; 100× yeastolate solution; 100× lactalbu-

and Maintenance min hydrolysate solution; 100× antibiotic/antimycotic solu-
of Sf9 Cells tion; fetal bovine serum (see Note 1).
 Purification of CDK7, CDK8 and CDK9 15

Clone ORFs in plasmids

Co-transfect plasmid with

AcNPV DNA in Sf9 cells
OR
Transform E.coli DH10Bac
cells with transfer plasmid,
select colonies and isolate
recombinant AcNPV DNA.
Transfect Sf9 cells.

Establish Passage 1 virus

stocks. Freeze aliquots.

Produce large volumes of Co-infect large culture

high titer virus stocks. of Sf9 cells at MOI=4.
(108 pfu/ml) Prepare cell extract.
2-4 weeks 2 days

Purify the recombinant

CDKs on Ni-NTA
beads.

Immediately exchange
buffer in PD10 columns

Purify the recombinant

kinases on MonoS
beads
1 day

Fig. 1 An outline of the expression and purification of CDK complexes. Cloning of

the proteins of interest in transfer plasmids and the production of recombinant
AcNPV usually takes 2–4 weeks. The subsequent expression of the kinases takes
2 days. The purification of the kinases over Ni-NTA and MonoS resins should be
performed in a single day

2. Tissue culture hood.

3. 28 °C incubator.
4. Tissue culture flasks (25, 175 cm2).

2.2 Expression 1. Cell lysis buffer: 10 mM Tris–HCl pH 7.5, 10 mM NaCl,

and Purification 2 mM β-mercaptoethanol, 0.5 mM EDTA, 10 mM
of Recombinant 2-glycerophosphate, 0.5 mM Na-vanadate Na3VO4, 2 mM
Kinases NaF. Add 2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml
pepstatin (see Note 2), 0.2 % (v/v) NP-40. Add 50 μg/ml
PMSF immediately before use.
2. 5 M NaCl.
3. 80 % glycerol.
16 Reena Pinhero and Krassimir Yankulov

4. 0.5 M imidazole–HCl pH 7.6.

5. Dounce homogenizer, 40 ml.
6. SW50.1 rotor and a compatible ultracentrifuge.

2.3 Purification 1. Ni2+-NTA Agarose.

of CDK Complexes 2. Equilibration buffer: 10 mM Tris–HCl pH 7.6, 0.5 M NaCl,
by Ni2+-NTA 5 mM imidazole, 10 % (v/v) glycerol. Add 50 μg/ml PMSF
immediately before use.
3. Ni-NTA Elution buffer: 10 mM Tris–HCl pH 7.6, 100 mM
NaCl, 10 % (v/v) glycerol. Supplement with 5, 15, 25, 100,
and 400 mM imidazole–HCl as indicated. Add 50 μg/ml
PMSF immediately before use.
4. Disposable 10 ml columns.

2.4 Purification 1. PD10 buffer exchange columns.

of CDK Complexes 2. 5 ml Econo-Pac Mono S cartridge.
by Mono S
3. S-buffer: 25 mM Hepes.Na pH 7.6, 0.1 mM EDTA, 1 mM
Chromatography
DTT, 5 % (v/v) glycerol, supplemented with 80 or 500 mM
NaCl. Add 50 μg/ml PMSF immediately before use.

2.5 Expression 1. Competent E. coli BL21(DE3)lysS cells.

and Purification 2. LB (Luria Broth)/amp plates and LB/amp liquid medium.
of GST-CTD Substrate
3. Bacterial shaker/incubator set at 37 °C.
4. IPTG powder.
5. TEN buffer: 20 mM Tris–HCl pH 7.5, 5 mM EDTA, 200 mM
NaCl. Add 50 μg/ml PMSF before use.
6. Protease inhibitors (pepstatin, leupeptin, aprotinin) at 1 mg/ml
(see Note 2).
7. Glutathione-agarose beads.
8. 1 M glutathione reduced.
9. 10 % Triton X-100 (TX100).
10. Disposable 10 ml columns.
11. One 10 % SDS-PAGE gel.
12. A mid-speed centrifuge (Sorvall Evolution RC or similar),
rotors (Sorvall SA300, SLA1500 or similar), 250 ml centri-
fuge bottles, 35 ml centrifuge tubes.
13. A sonicator, Misonix XL or similar.

2.6 Kinase Assays 1. Kinase buffer: 20 mM Tris–HCl, pH 8, 50 mM KCl, 7 mM

MgCl2, 5 mM 2-glycerophosphate, 100 μg/ml BSA, 10 μM
ATP, 2 μCi (7.4 × 104 Bq) α-32P-ATP.
2. 1 mg/ml GST-CTD.
3. 1 mg/ml myelin basic protein (MBP).
 Purification of CDK7, CDK8 and CDK9 17

2.7 Other Equipment 1. Basic radiation safety equipment and Geiger counter.
2. Mini-PROTEAN II electrophoresis cell (Bio-Rad) or compa-
rable mini-gel system.
3. Microcentrifuge, standard molecular biology equipment.

3 Methods

3.1 Production The viruses for expression of CDK7, MAT1 [17] and CycC [18]
of and Handling were gifts from Dr. D. Morgan and Dr. E. Lees, respectively. The
of Recombinant virus for the expression of His6-CDK9 and CycT1 was produced
Viruses by pBAC4/CDK9/CycT1 plasmid [19] and the BacVector-3000
kit (Novagen) according to the instructions of the manufacturer
(see Note 3). The virus for the expression of His6-CycH was pro-
duced by cloning CycH into pBlueBac and the Bac-N-Blue trans-
fection system (Invitrogen Life Technologies) (see Note 3). The
recombinant virus was purified through three rounds of selection
of blue plaques. His6-CDK8 was produced by cloning CDK8 into
pFastBacHTa and the BAC-to-BAC recombination system
(Invitrogen Life Technologies) according to the instructions of the
manufacturer (see Note 3). All three systems are reliable and pro-
duce viruses with equal success. Space limitations preclude exten-
sive details on these procedures. However, we must note that in
our hands Bac-to-Bac (Invitrogen Life Technologies) has been
most user-friendly and least time-consuming.

3.2 Amplification The initial preparations of recombinant baculovirus (termed

of Recombinant Passage 1 virus stocks) typically contain low titer of the virus. For
Viruses the viruses produced by the Bac-N-Blue system Passage 1 corre-
sponds to the cell culture infected with a virus plaque that has been
purified by three rounds of plaque selection. Passage 1 for the
viruses produced by Bac-to-Bac or BacVector systems is the trans-
fected Sf9 cultures themselves. High titer viral stocks are prepared
from Passage 1 by two-step amplification as described below.
1. Infect 10 ml Sf9 cells at 0.1–0.3 × 106 cells/ml in a 25 cm2
T-flask with 0.1 ml of virus Passage 1. The expected Multiplicity
of Infection (MOI) is in the range of 0.05–1 (see Note 4).
2. Incubate for 4–6 days at 28 °C. At this point all cells should be
in suspension (see Note 5).
3. Spin the culture at 275 × g for 5 min in sterile 15 ml conical
tubes and collect the supernatant. These supernatants contain
1 × 106–1 × 107 plaque forming units per ml (pfu/ml).
4. Freeze 1 ml aliquots as Passage 2 and store at −80 °C
(see Note 6).
5. Infect 75 ml Sf9 cells at 0.5–1 × 106 cells/ml in a 175 cm2 flask
(Sarsted) with 1 ml of the passage 2 stock. Incubate for 4 days
18 Reena Pinhero and Krassimir Yankulov

and collect the supernatant. These supernatants typically con-

tain 1 × 108 pfu/ml.
6. Use the supernatants from step 5 for large-scale protein
expression. The viral stocks can be stored at 4 °C for several
weeks.

3.3 Infection High levels of expression are achieved by the infection of high den-
and Harvesting sity Sf9 cells at high MOI. The gain of yields exceeds the concerns
of Insect Cells of superfluous virus recombination.
1. Grow Sf9 cells to 1.5–2 × 106 cells/ml in several (5–8) 175 cm2
T-flasks or in a roller-flask.
2. Infect each flask with 2 ml of passage 4 of each of the appro-
priate viruses (MOI = 4) and incubate for 48 h at 28 °C
(see Note 7). All cells must be floating (100 % infection) 30 h
post infection. If less than 100 % infection is observed, do
NOT proceed. Significantly lower yields of recombinant pro-
tein should be expected.
3. Harvest the cells after 48 h by spinning at 275 × g for 5 min at
4 °C.
4. Pour off the supernatant, loosen the pellet by gently shaking
it, and wash the pellets with 15 ml of chilled phosphate buff-
ered saline.

3.4 Preparation Cell lysis and chromatography must be performed on ice with pre-
of Cell Lysate chilled buffers. Samples should be constantly kept on ice or in a
4 °C cold rooms.
1. Resuspend the cells in 3 pellet volumes of Cell lysis buffer. At
this point the cells can be immediately processed or frozen at
−80 °C (see Note 8).
2. Transfer the cell lysate to a Dounce homogenizer and break
cells by ten strokes. Check for cell lysis under a microscope.
3. Add imidazole and NaCl to a final concentration of 5 mM and
0.5 M, respectively. Homogenize by two additional strokes.
4. Transfer the cell lysate to a 50 ml screw cap tube. Rock for
30 min in a cold room.
5. Spin at 75,000 × g at 4 °C in SW50.1 rotor for 30 min.
6. Transfer the supernatant to a fresh prechilled tube; add glyc-
erol and MgCl2 to final concentrations of 10 % and 3 mM,
respectively (see Note 9).

3.5 Ni2+-NTA The Ni2+-NTA and Mono S chromatography steps should be per-
Chromatography formed without any freezing and thawing of the samples. Buffer
exchange should be in desalting columns rather than by dialysis
(see Note 10). Several 10 % SDS–polyacrylamide gels should be
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investment industry employees. 25th ed. 68 p. © New York Institute
of Finance; 28Feb66; A835996. NEW YORK JURISPRUDENCE. ©
Lawyers Co-Operative Pub. Co. Vol. 45. © 21Feb66; A817946. 46, ©
25Mar66; A828023, 47, © 22Apr66; A836941, NEW YORK
JURISPRUDENCE. 1966 cumulative supplement. Rochester, N.Y.,
Lawyers Co-operative Pub. Co. 40 v. © Lawyers Co-operative Pub.
Co.; 10Jan66; A811508. NEW YORK LAW OF LANDLORD AND
TENANT. 1965 cumulative annual pocket part. Brooklyn, E.
Thompson Co. 3v. Appl. authors: West Pub. Co. & Edward Thompson
Co., employers for hire. © West Pub. Co.; 27Dec65; A811776. NEW
YORK LAW OF WILLS. Supplement. Brooklyn, E. Thompson Co. 2v.
Appl. authors: West Pub. Co. & Edward Thompson Co., employers
for hire. © West Pub. Co.; 30Dec65; A812215. 1965 cumulative
NEW YORK LAWYERS DIARY AND MANUAL; bar directory of the
State of New York, 1966. Newark, N.J., New York Lawyers Diary &
Manual. 976 p. © New York Lawyers Diary & Manual; 13Dec65;
A809488. NEW YORK LEGISLATIVE SERVICE, INC. See NEW aos
STATE LEGISLATIVE ANNUAL, 1965. NEW YORK LIFE INSURANCE
CO. A brief ovtline of medicare. Sheet. © New York Life Insurance
Co.; 23Mar66; A837293. Business health insurance primer for field
underwriters. See NEW YORK LIFE INSURANCE CO. HEALTH
INSURANCE SALES DIVISION. 721 Canada health insurance field
rate book. 1v. Appl. author: Lowell M. Dorn. © New York Life
Insurance Co.; 25Apr66; A837579. Charity and the family budget.
DAVID, LESTER, Field rate book, Canada & Supplement, Jan., 1966.
2v. Appl. author: Lowell M, Dorn. © New York Life Insurance Co.;
lJan66; AG23448, Field rate book, United States, female lives &
Supplement. 2 v. Appl. author: Lowell M, Dorn, NM: additions &
revisions. © New York Life Insurance Co.; lJan66; A827822, Field
rate book, United States, male lives & Supplement, Jan., 1966. 2v.
Appl. author: Lowell M. Dorn, © New York Life Insurance Co.;
lJan66; AS23449. The importance of your company benefits. See
DAVID, LESTER. Pension plan manual, United States. 48 p. Appl.
author: Lowell M. Dorn. © New York Life Insurance Co.; lJan66;
AS44847. Pension trust and profit-sharing Service manual. 1v. ©
New York Life Insurance Co.; 25Feb66; A838119. Proceedings of the
64th annual managers! me€ting ... January 2530, 1966, Hollywood
Beach, Florida. 171 p. © New York Life Insurance Co.; 15dun66;
A844149. Should you go into business for yourself. See CROSS,
WILBUR. See BELTING, See The taxes you pay: CHARLES E. United
States health insurance field rate book; men and women. 1 v, Appl.
author: Lowell M. Dorn. © New York Life Insurance Co.; 25Apr66;
A837578. NEW YORK LIFE INSURANCE CO, INSURANCE SALES
DIVISION. Business health insurance primer for field underwriters.
24 p. © New York Life Insurance Co.; 11Mar66; A843683. HEALTH
NEW YORK SOCIETY OF ARCHITECTS. Manual, 1966, New York
building laws. 54th ed. 800 p. © New York. Society of Architects;
3Jan66 (in notice: 1965); A822971. NEW YORK STATE BAR ASSN.
LAW SECTION. Antitrust law symposium, 1966. New York,
Commerce Clearing House. 185 p. © Commerce Clearing House,
Inc.; 11May66; A844581. ANTITRUST NEW YORK STATE
FEDERATION OF OFFICIAL PLANNING ORGANIZATIONS. Planning,
zoning & subdivision. See ANDERSON, ROBERT M. NEW YORK
STATE INDUSTRIAL DIRECTORY, INC. See NEW Bae STATE
INDUSTRIAL DIRECTORY, 1966. NEW YORK STATE INDUSTRIAL
DIRECTORY, 1966. 820 p. © New York State Industrial Directory,
Inc.; 3Feb66; A817658. NEW YORK STATE LEGISLATIVE ANNUAL,
1965. A. Fairfield Dana, editor, New York, New York Legislative
Service. 667 p. © New York Legislative Service, Inc,; 30Dec65;
A831659. These entries alone may not reflect the complete
Copyright Office record pertaining to a particular work. Contact the
U.S. Copyright Office for information about any additional records
that may exist.
 722 BOOKS Jan.—June NEW YORK STATE SCHOOL
BOARDS ASSN. , Baldwinsville telephone directory, Canastota
telephone directory. INC, 1966-67. © 13Apr66; A834310. 1966-67.
© l4Jun66; AS43324, School insurance. See ROGERS, Ballston Spa
telephone directory. Carthage special telephone directory. E. LLOYD,
1965-1966, © 227un65; AB1B10H., 5Dec65. © 24Nov; A818080.
NEW YORK STOCK EXCHANGE, An introduction to financial
statement accounting, STOCK EXCHANGE, FIRMS LIAISON, See
NEW YORK DEPT, OF MEMBER Preliminary issue of Stocks on the big
board. May 1966. Statistical material compiled by Standard & Poor's
Corp. Rev. 31 p. © New York Stock Exchange; 15Mar66; Barker,
Lyndonville telephone directory. 1966. © 21Jan66; A819153.
Barneveld telephone directory. 1965-1966. © 260ct65; a818103.
Batavia telephone directory. 1966. © 3lJan; A819138. Bath
telephone directory. 1966. © 17Dec65; A819144. Carthage
telephone directory. 1966-67. © 13May66; A843325. Castleton
telephone directory. 1966. © 15Dec65; A819177. Catskill, Palenville
telephone directory. 1966-67. © 8Jun66; A8433518, Cazenovia
telephone directory, A828938 . Beacon, Fishkill telephone direc-
1966-1967. © 13Apr66; A834556. Cory» Central Bridge, Cobleskill
Study outline for member, allied 1965-1966. © 20Nov65; A818116.
See 2 Study plan, NEW YORK STOCK EXCHANGE. member and
coordinate examinations. 12 p. NM: revisions & additions. © New
York Stock Exchange; 31Dec65; A831054. ed ed. 13p. © New York
Stock Exchange; 2Feb66; A828761. DEPT. OF MEMBER FIRMS
LIAISON, An introduction to financial stateBolivar, Cuba telephone
directory. 1966-67. © 7Apr66; A834313. Bronx address telephone
directory. © ZNov65; A808749. © 2Dec65; A816602. © 4Jan66;
A811579. © 7Feb66; A816979. © 10Mar66; A825723. © 11Apr66;
A843314, © 18May66; A838615. The Bronx telephone directory.
Richmondville, Sharon Springs, telephone directory. 1965-1966. ©
280ct65; A818075. Central Bridge special telephone directory, ©
30ct65; A8Z43526. Chittenango, Bridgeport telephone directory.
1966-67. © 13Apr66; A834319, Cicero telephone directory. 1966-67.
© 123Apr66; A8343518. land, Constantia telephone ment
accounting. 2ded. 28 p. 1966. © 14Feb; A816060. eee i e eotneee
Stock Exchange; 31Dec65; The Bronx telephone directory. 1966-67.
© 13Apr66; A834317. NEW YORK SUPPLEMENT, SECOND SERINS.
[With one star on bolt 1966-67. © l4Feb66; eee Cold Spring Harbor,
Commack, Huntington, and others yellow The Bronx yellow pages.
The red pages, The red book. [Coded © West Pub. Co. Vol. book.
HS-C ] 263. © l4Feb66; A817574. 1966. © l4Feb; 4816598. 1966-
67. © 2Jun66; A844520, 264. © 30Mar66; A830038. 265. ©
29Apr66; Heese 266, © 27May66; A8 4720, Brooklyn address
telephone directory. © 17Nov65; A811110, © 20Dec65; A814740.
Colonie telephone directory. 1966. © 15Dec65; A819142.
Cooperstown, Cherry Valley, Edmeston, and others telephone NEW
YORK TSLEPHONE CO. © 25Jan66; A816982. directory. Heading list,
See DONNELLEY © ZMar66; A825724. 1965-1966. © 28Jun65;
A818065. (REUBEN H.) TELEPHONE DIRECTORY © 31Mar66;
A850122. [Telephone directories] co. © New York Telephone Co,
Albany street address telephone directory. © 1Ju165; A818122, ©
27Apr66; AS44811, Brooklyn telephone directory. 1966. © 14mMar;
A827797. Brooklyn telephone directory. [With one star on bolt]
Corning, Campbell, Caton and others telephone directory. 1966. ©
15Dec65; A819165. Cornwall telephone directory. 1966. © 26Jan66;
A819164. Dunkirk telephone directo © 6Aug65; A818121 1966-67.
© 14Mar66; A825720, 1965-1966. © 270ct65; ABSH328 © 2Sep65;
A818137. Brooklyn yellow pages. The red East Berne,
Rensselaerville, West © 60ct65; A818129, book. Berne, Westerlo
telephone di© 1Nov65; A818127. 1966. © 14Mar; A827796. rectory.
© 9Dec65; A819191. 1966. © 15Dec65; A819182. © 12Jan66;
A819190. © 15Feb66; A819185. © 18Mar66; A834367. © 25Apr66;
A834369. © 25May66; AS44842, Albany telephone directory. 1966.
© 15Dec65; A819176. Albion telephone directory. 1966. © 10Jan;
A819139. Alexandria Bay, Clayton, Thousand Islands telephone
directory. 1966-67. © 13May663 A843351. Altamont, Clarksville,
South Bethlehem, Voorheesville telephone directory. Broome County
telephone directory with yellow pages. Binghamton SC. 1966-67. ©
26May66; A843320, Broome County telephone directory With yellow
pages. Endicott ed. 1966-67. © 24May66; AS45346. Buffalo street
address telephone directory. © 30Jun65; A818125. © 30Jun65;
A818126, © 233u165; A818131, © 14Sep65; A818138, © 150ct65;
A834381, © 26Nov65; A834368, © Bapecee ABLOLSS. East
Syracuse telephone directory. 1966-67. © 13Apr66; A834304.
Eastern Suffolk yellow pages. The red book, [Coded RP-C]. 1966-67.
© 8Jun66; A8+4519. Ellicottville, Little Valley telePhone directory.
1966-67. © 6Apr66; A834365. Elmira, Horseheads telephone
directory. 1966. © 7Jan66; A819158. Esperance special telephone
directory. © 30ct65; A818074. A soeait s Fayetteville, Minoa
telephone 1966. © 15Dec65; A819181, 2 oa (in notice: 1965);
directory, Amityville, Babylon, Bay Shore, © 25Feb66 (in notice:
1965); Beo-Gi- a 1 7Her Ges HOA: and others yellow pages, The
AB34374. Fleischmanns , Pine Hill telephone red book, [Coded BB-C]
© 28Mar66 (in notice: 1965); directory. 1966-67. © 3Jun66;
Agh45e1, A834375. 1965-1966. © 2kJun65; A834335. Amsterdam
telephone directory. © 27Apr66; A834376, © 2iayoo, ABLUGA
Fleischmanns special telephone Bo -67. 11May66; A843322.
directory. Be HENS cue ae Cairo telephone directory, 5Dec65. Ye
24Nov; A818068. Arcade, Bliss, Chaffee, and others 1966-67, ©
8Jun66; A843336, telephone directory. Cambridge, Salem telephone
direcFranklinville special telephone 1965-1966. © 6Aug65; A818069.
directory. | tory. © 15Aug65; a818079. 4 Attica, Varysburg
telephone 1965-1966. © 22Jun65; A818100. 3 i directory. Camden
telephone directory Franklinville telephone directory. iy 1966. ©
31Jan; A819137, : 1966-67. 1966-67. © 14dun66; A843360.
Camillus telephone directory. 1966-67. © 13Apr66; A834312. ©
6Apr66; A824361. mS Freehold, Greenville, Oak Hill telephone
directory, 1966-67. © 8Jun66; A843337. Auburn telephone directory.
1966-67. © 7Jun66; A843342, Le LT a a IRN ses Oe: a see . See y ,
ieee These entries alone may not reflect the complete Copyright
Office record pertaining to a [DeAEe JEL work. Contact the U.S.
Copyright Office for information about any additional records that
may exist.
 1966 CURRENT REGISTRATIONS Gasport telephone
directory. 1966. © 21Jan66; A819156. Geneva, MacDougall
telephone direcory. 1965-1966. © 22Ju165; A834331. Glens Falls
telephone directory. 1965-1966. © 15Jul65; A818111. Gouverneur
telephone directory. 1965-1966. © 28Ju165; A818088. Gowanda
telephone directory. 1965-1966. © 9Nov65; A834327. Granville
telephone directory. 1965-1966. © 15Ju165; A818105. Greenwich,
Schuylerville telephone directory. 1965-1966. © 22Jun65; A818099.
Greenwood Lake telephone directory. 1966. © 26Jan; A814739.
Groton, McLean telephone directory. 1965-1966. © 4Nov65;
A834334. Hamilton telephone directory. 1965-1966. © 10ct65;
4818071. High Falls, Rosendale telephcne directory. 1966-67. ©
21Apr66; A8Z4358. Highland, Clintondale telephone directory. 1965-
1966. © 24Nov65; A818094. Highland Falls telephone directory.
1966. © 26Jan; A816599. Holley telephone directory. 1966. ©
10Jan; A819140. Hoosick Falls telephone directory. 1966-67. ©
2May66; A854315. Hornell telephone directory. 1966. © 16Dec65;
A819143. Hudson, Claverack telephone directory. 1966-67. ©
8Jun66; A843326, Hunter special telephone directory. 16Jan66. ©
16Jan; A819150. Hunter telephone directory. 1966-67. © 8Jun66;
A843340. Ilion, Frankfort telephone direcory. 1965-1966. ©
11Nov65; A818106. Ithaca, Lansing and Newfield telephone
directory. 1965-1966. © 1Nov65; A818093. Jewett, Lexington,
Prattsville, Windham special telephone directory. 16Jan66. © 16Jan;
A819162. Jewett, Lexington, Prattsville, Windham telephone
directory. 1966-67. © 8Jun66; AS43544. Jordan telephone directory.
1966-67. © 13Apr66; a834302. Kingston, Esopus, Shokan telephone
directory. 1966-67. © 21Apr66; A834360. Lake Placid, Saranac Lake,
Tupper Lake telephone directory. 1966-67. © 10May66; A843315, ‘
Lewiston telephone directory. 1966-67. © 3Mar66; A834349. a Little
Falls, Dolgeville, Salisbury Center telephone directory. ; - 1965-1966.
© 11Nov65; A818102, ! Liverpool telephone directory. : 1966-67. ©
13Apr66; A834303. Lockport telephone directory. 1966. © 2lJan66;
A819161. Malone telephone Sore cr ORRe 1965-1966, © 8Jul65;
A3834325, Manhattan address telephone directory. © 23Nov65;
A811109. © 23Dec65; AS14741. © 19Jan66; A816981. © 16Feb66;
A825725. © 15Mar66; A830121, © 14Apr66; A838385. © 10May66;
A838614, Manhattan yellow pages. The read book, 1966. © 7Jan;
A810930. Manlius telephone directory, 1966-1967. © 13Apr66;
A834353. Marlboro telephone directory. 1966. © 26Jan66; A819163.
Massena telephone directory. 1965-1966. © 28Ju165; A818108.
Mechanicville telephone directory. 1966-67. © 5May66; A843352.
Medina telephone directory. 1966. © 2l1Jan66; A819155. Mexico,
Parish telephone directory. 1966-67. © 10Mar66; A834316.
Middleport telephone directory. 1966. © 21Jan66; A819154. Milton
telephone directory. 1965-1966. © 2'Nov65; A818087. Moravia
telephone directory. 1966-67. © 7Jun66; A843327. Munnsville
telephone directory. 1966-1967. © 14Jun66; 4843350. New Paltz
telephone directory. 1965-1966. © 24Nov65; A818089. Newburgh
telephone directory. 1966. © 25Jan66; A819160. Newfane telephone
directory. 1966. © 2l1Jan66; A819159. Niagara Falls telephone
directory. 1966-67, © 3Mar66; AS34341. North Syracuse telephone
directory. 1966-67. © 13Apr66; A834309. Cec SpeGuEs telephone
directory. 1965-1966. © 283u165; A818078. Olean, Allegany,
Hinsdale, Portville telephone directory. 1966-67. © 6Apr66;
A834364, Oneida, Sherrill, Silvan Beach telephone directory. 1966-
67. © 14Jun66; A843328, Oneonta, Davenport, Otego, and others,
telephone directory. 1965-1966. © 28Jun65; 1831530. Oswego
telephone directory. 1966-67. © 27Apr66; A854314. Owego, Nichols,
Tioga Center telePhone directory. 1966-67. © 24May66; A843332.
Pawling, North Clove, Patterson, Wingdale telephone directory. 1965-
1966. © 2\'Nov65; A818096. Penn Yan telephone directory. 1965-
1966. © 223u165; A834321. Phelps and Clifton Springs, N.Y.,
telephone directory. See ONTARIO TELEPHONE CO., INC. [Telephone
directories] Philmont telephone directory. 1966-67. © 8Jun66;
A843331. Phoenicia telephone directory. 1966-67. © 21Apr66;
A854350. Plattsburgh telephone directory. 1965-1966. © 153u165;
A818114. Port Byron telephone directory. 1966-1967. © 7Jun66;
A843333. Port Henry, Ticonderoga, Hague, and others telephone
directory. 1965-1966. © 16Jun65; A818073. Potsdam, Madrid,
Norfolk, and others telephone directory. 1965-1966. © 28Ju165;
A818110. 723 Queens address telephone directory. © 9Nov65;
A811107. © 7Dec65; A811106. © 6Jan66; AS1474e. © 9Feb66;
A816980. © 1Mar66; A825726. © 6Apr66; A831086, © 5May66;
A838386. © 2Jun66; A844812, Queens telephone directory. 1966-
67. © l14Apr66; A831085. Queens yellow pages. The Red book,
1966-67. © 14Apr66; A830120. Ransomville telephone directory.
1966-67. © 3Mar66; AS34340. Rensselaer telephone directory. 1966.
© 15Dec65; A819180. Richfield Springs special telephone directory.
© 19Sep65; A818081. Rockaways telephone directory. Nassau ed.
1966-67. © 25Apr66; A831084. Rockaways telephone directory.
Queens ed. 1966-67. © 25Apr66; A831083. Rome telephone
directory. 1966-67. © 14Jun66; A845347. Salamanca, Limestone
telephone directory. 1966-67. © 6Apr66; A834338. Saratoga Springs
telephone directory. 1965-1966. © 22Jun65; A818101. Saugerties
telephone directory. 1966-67. © 21Apr66; A834362. Schenectady
street address telephone directory. © 67u165; A818135. ©
10Aug65; A818128. © 14Sep65; A818134. © 210ct65; A818132. ©
23Nov653; A854380. © 43Jan66; A819185. © 16Feb66; A819186.
© 18Mar66; A834370. © 14Apr66; A834372. Schenectady
telephone directory. 1966-67. © 18Feb66; A819175. Schroon Lake
special telephone directory. © 7Nov65; A819141. Seneca Falls,
Waterloo, Fayette, telephone directory, 1965-1966. © 22Jul65;
A818070. Silver Creek, Forestville, telephone directory. 1965-1966.
© 270ct65; A818072. Skaneateles telephone directory. 1966-67. ©
13Apr66; A834311. Springville telephone directory, 1965-1966. ©
9Nov65; A834329, Stamford, Grand Gorge, Hobart, and others
telephone directory. 1965-66. © 28Jun65; A834520. Staten Island
address telephone directory, © 29Nov65; A811105, © 13Jan66;
A820035, © 27Jan66; A816603, © 2l\Feb66; A825722, © 2l\Mar66;
A83838),. © 21Apr66; A833770. © 19May66; A838613, Staten
Island telephone directory. [With two dots on bolt] 1966-1967. ©
3Dec65; A816601. Staten Island telephone directory. (With two stars
on bolt] 1966. © 3Dec65; A811104, pn eh a These entries alone
may not reflect the complete Copyright Office record pertaining to a
particular work. Contact the U.S. Copyright Office for information
about any additional records that may exist.
 Secunia alia 724 NEW YORK TELEPHONE CO.-Con.
[Telephone directories] -Con. Suffolk telephone directory. [With one
star on bolt] 1966-67. © 2Jun66; A844518, Suffolk telephone
directory. With yellow pages, coded BB. 1966-67. © 3Jun66;
A839992. Suffolk telephone directory. With yellow pages, coded HS.
1966-67. © 2Jun66; A839993. Suffolk telephone directory & Yee aea
pages. [Coded RP] 1966-67 © 8Jun66; A8B41613. Syracuse
metropolitan area telephone directory. 1966-67. © on66: ABZAS47.
Syracuse street address telephone directory. © | icemne 3; ACI8120.
BO0Sep65; AB1E136. 280ct65; A818130. 2Dec65; A819188 6Jano6;
AS19184, 28Jan66; A819187, 28Feb66; A834366, 15Apr66;
AS34373, 12May66; ABYUBLO, Tannersville, Haines Falls special
telephone directory. 16Jan66. © 16Jan; A819151. Tannersville,
Haines Falls telephone directory. 1966-67. © 8Jun66; A843543. Troy,
Cohoes, Waterford, Watervliet street address directory. 7Jul65;
A818133. 11Aug65; A818124. 9Sep65; A818123. 210ct65; A818119.
24Nove5; ASS4377. 30Dec65 (in notice: 1966); A834379. © 9Feb66;
A854378. © 21Mar66; AG34371. © 2May66; A844798, © ZlMay66;
AS44841, Troy telephone directory. 1966-67. © 5May66; AG4 3354,
Tully, Amber, Fabius, La Fayette telephone directory. 1966-67. ©
13Apr66; A834308. OXOKOKONG ae OOOOOO Vernon, including
Rome and Oneida, N.Y., telephone directory. See VERNON
TELEPHONE CO. [Telephone directories] Wappingers Falls telephone
directory. 1965. 1966. © 24Nov65; A818107. Watertown telephone
directory. 1966-67. © 13May66; A843323. Watkins Glen, Montour
Falls telephone directory. 1966. © 7Jan66; A819157. Waverly
telephone directory. 1965-1966. © 29Nov65; Ad19145. Wayne
telephone directory. Newark ed. 1966-67. © 25Mar66; A8Z4s4e,
Wayne telephone directory. Sodus ed. 1966-67. © 25Mar66;
A8343548. Wellsville telephone directory. 1966-67. © 7Apr66;
A834301. Westchester address telephone directory. © 16Nov65;
A811108. © 21Dec65; A811580. © 31Jan66; A816604, © 8Mar66;
A825721. © 8Apr66; A835740. © 12May66; A838616, ee BOOKS
Westchester, Putnam telephone directory, 1966. on bolt] PA. ©
10Nov65; A816600. Westchester, Putnam, telephone directory with
yellow pages for your locality, K. on bolt. 1966. © 10Nov65;
A808748. Whitehall poraenone directory. 1965-1966. © 15Ju165;
818077 Wilson telephone directory. 1966. © 2lJan; A854335.
Woodstock telephone directory. 1966-67. © 21Apr66; A8354339.
Youngstown telephone directory. 1966-67. © 3Mar66; A834363,
Yellow pages heading list, downstate New York. See DONNELLEY
(REUBEN H. ) TELEPHONE DIRECTORY CO. [Telephone directories]
See C & U TELEPHONE CORP, CAPE VINCENT TELEPHONE CO.
CHAMPLAIN TELEPHONE CO, CHAZY & WESTPORT TELEPHONE
CORP. COLUMBIA & RENSSELAER TELEPHONE CORP, CROWN POINT
TELEPHONE CORP. DELHI TELEPHONE CO. — DEPOSIT TELEPHONE
CO. DIMOCK HOLLOW TELEPHONE CO, EMPIRE TELEPHONE CORP.
FINGER LAKES TELEPHONE CORP. GERMANTOWN TELEPHONE CO.,
INC. HANCOCK TELEPHONE CO. HIGHLAND TELEPHONE CO,
TROQUOIS TELEPHONE CORP. MID-STATE TELEPHONE CO,, INC.
NEWPORT TELEPHONE CO., INC, NICHOLVILLE TELEPHONE CO.,
INC. ODESSA TELEPHONE CO, ONEIDA COUNTY RURAL TELEPHONE
CO, OSWEGO COUNTY TELEPHONE CORP. PATTERSONVILLE
TELEPHONE CO, PORT BYRON TELEPHONE CO, RED HOOK
TELEPHONE CO. SOUTHERN TIER TELEPHONE co. SOUTHERN TIER
TELEPHONE CO., INC. STATE TELEPHONE CO, SYLVAN LAKE
TELEPHONE CO., INC, TOWNSHIP TELEPHONE CO, TRUMANSBURG
HOME TELEPHONE CO. WALDEN TELEPHONE CO, WARWICK
VALLEY TELEPHONE CO, WESTERN COUNTIES TELEPHONE CORP,
WESTERN COUNTIES TELEPHONE CORP. MARATHON DIVISION.
THE NEW YORK TIMES. A century of great inventions. With an
introd. by Walter Sullivan. lv. (A Current affairs publication of the
Office of Educational Activities) NM: compilation of text & illus. ©
New York Times Co.; 13Dec65; A807900. Communist China; giant of
the Orient. By Seymour Topping. 15 p. (A Current affairs publication
of the Office of Educational Activities) NM: compilation. © New York
Times Co.; 15Mar66; A839061. Crosswords from the Times; daily
puzzles. Series 14. Compiled & edited by Margaret Farrar. New York,
Simon & Schuster. 1 v. NM: compilation & foreword. © The New
York Times; 15May66; AS84453. [With two stars With two stars Jan.
—June Essays from the New York times. With an introd. by Theodore
M. Bernstein. 1v. (A Current affairs publication of the Office of
Educational Activities) NM: text & compilation of illus. © New York
Times Co.; 26Jan66; A839072. Here is a miniature reproduction of
last year's New York times summer theater preview. 1 v. NM:
compidation of illus. © New York Times Co.; 17May66; 4839073.
Hiroshima plus 20. Introd. by John W. Finney. New York, Delacorte
Press. 211 p. NM: editorial compilation & revision. © New York
Times Co.; 280ct65; A809436. Index. John Rothman, editor. New
York, Reprinted for the New York Times Co. by R. R. Bowker Co. NM:
forewords. © New York Times Co. Vol. no. 1, no.1-2, © 11lAug65;
A809054. 1, no.3-4+. © 11Aug65; A809056. 2, no.1-2. © 11Aug65;
A809057. 2, no.3-4+. © 11Aug65; A809061. 3, no.1-2. © 11Aug65;
A809062. 3, no.3-4. © 1lAug65; A809063. 4, no.1-2. © 11Aug65;
A809055. 4, no.3-4. © 11Aug65; A809064. 5, no.1-2. © 11Aug65;
A809070. 5, no.3-4. © 1lAug65; A809058. 6, no.1-2. © 11Aug65;
A809059. 6, no.3-4. © 11Aug65; A809060. 7, no.1-2. © 11Aug65;
A809065. 7, no.3-4+. © 1lAug65; A809066. 8, no.1-2. © 20Jul65;
A809067. 8, no.3-4+. © 20Jul65; A809068. 9, no.1-2. © 20Jul65;
A809069. The issues facing Americans. With commentary on
international affairs by Max Frankel. National affairs by A. H, Raskin.
23 p. (A Current affairs publication of the Office of Educational
Activities) © New York Times Co.; 7Feb66; A839068. The
Mediterranean. By Robert C. Doty. 19 p. (A Current affairs
publication of the Office of Educational Activities) NM: text &
compilation of illus. © New York Times Co.; 11May66; A839075. The
more abundant life. 15 p. (A Current affairs publication of the Office
of Educational Activities) NM: compilation of text & illus. © New York
Times Co.; 29Nov65; A807899. The night the lights went out. Edited
by A. M. Rosenthal & Arthur Gelb. New York, New American Library.
158 p. (A Signet book, T2853) Appl. states: rewritten & expanded
from articles in the New York times. © New York Times Co.;
29Nov65; A812339. Portrait of a decade. ANTHONY . The U.S.
judiciary. By Fred P. Graham. 14 p. (A Current affairs publication of
the Office of Educational Activities) NM: compilation of illus. © New
York Times Co.; 18Apr66; A8359062. See LEWIS, World affairs
workshop. See ENCYCLOPAEDIA BRITANNICA PRESS, INC. World
economic review and forecast, 1966. Edited by Lee Kanner, With an
appraisal of the economy by Thomas E, Mullaney. New York, Grosset
& Dunlap, 256 p. © New York Times Co,; loreb66; A828951. These
entries alone may not reflect fire complete Conynent Office record
pereinine to a airarticula onl Contact the U.S. Copyright Office for
information about any additional records that may exist.
 1966 THE NEW YORK TIMES. TIONAL ACTIVITIES.
Introduction to a good reading habit. 37 p. (A Curriculum service
booklet NM: compilation. © New York Times Co.; 21Jul66; A853222,
OFFICE OF EDUCAIntroduction to a good reading habit 37 p. (A
Curriculum service booklet NM: partial textual & pictorial matter. ©
New York Times Co.; 7Jun66; A853225. NEW YORK TIMES CO.
Africa. See NIELSEN, WALDEMAR A. | A century of great inventions.
See THE NEW YORK TIMES, See THE NEW YORK Communist China.
TIMES, Essays from the New York times. See THE NEW YORK
TIMES, Furnishing the nursery, it's all child's play. See PLUMB,
BARBARA. Here is.a miniature reproduction of last year's New York
times summer theater preview. See THE NEW YORK TIMES.
Hiroshima plus 20. See THE NEW YORK TIMES. Index. See THE
NEW YORK TIMES. Introduction to a good reading habit. See THE
NEW YORK TIMES. EDUCATIONAL ACTIVITIES. The issues facing
Americans. See THE NEW YORK TIMES, Latin America. See SZULC,
TAD. OFFICE OF Mediterranean. See THE NEW YORK TIMES, The
more abundant life. See THE NEW YORK TIMES. The New York
times guide to dining out in New York. See CLAIBORNE, CRAIG. The
New York times guide to home furnishing. See PLUMB, BARBARA.
The New York times in distributive Education. See ENGLANDER,
FABIAN. New York times in the crowded curriculum. See WALSH,
PAUL L. The New York times in the economics elassroom. See
ROSTKER, MADELINE F. The New York times, versatile aid in Social
studies. See AUSTIN, LOUISE. The night the lights went out. See
THE NEW YORK TIMES, Public opinion. See FALON, BENJAMIN.
Southeast Asia. MAN. Speaking of books and life. See ADAMS, J.
DONALD. The U.S. judiciary, YORK TIMES, World affairs workshop.
Facts, Profiles, Activities & problems, By New York Times Co. &
Encyclopaedia Britannica Press, Inc. Cards. © New York Times Co. &
Encyclopaedia Britannica Press, Ine.; 29Nov65; A814910. World
economie review and forecast, 1966. See THE NEW YORK TIMES,
NEW YORK TIMES CO. See AMERICA'S CULTURAL REVOLUTION
(FILMSTRIP SCRIPT) COMMUNIST CHINA (FILMSTRIP SCRIPT)
FOCUS ON AFRICA (FILMSTRIP SCRIPT) See DURDIN, TILILSee THE
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