![78 Biotechnol. Prog., 2009, Vol. 25, No. 1
Figure 2. Western Blott analysis results of BGSC-1A751
(npr2, apr2) carrying pre(subC)::hGH and BGSC-
1A197 (spo2) carrying pre(subC)::hGH.
Lane 1, commercial (standard) hGH; Lane 2, hGH produced by
r-B. ‘subtilis BGSC-1A751 (nprÀ, aprÀ) carrying pMK4::pre
(subC)::hGH; Lane 3, hGH produced by r-B. subtilis BGSC-
1A197 (spoÀ) carrying pMK4::pre(subC)::hGH; and Lane 4,
marker (Sigma M 0671). Figure 3. SDS-PAGE analysis of rhGH, produced by r-B.subti-
lis BGSC-1A751 (npr2, apr2) carrying pMK4::
pre(subC)::hGH.
M, protein marker (Fermentas); Lane 1, product mixture of
and B. subtilis BGSC-1A197 (spoÀ) carrying pMK4::pre r-B.subtilis containing rhGH; Lane 2, 1st rhGH separation with
hGH specific aptamer; Lane 3, 2nd rhGH separation with hGH
(subC)::hGH were determined on a glucose (CG ¼ 6 g LÀ1) specific aptamer; and Lane 4, standard hGH.
based defined medium. The supernatant obtained by centrifuga-
tion at t ¼ 27 h of the fermentation was partially purified by
dead-end ultrafiltration and nearly 10-fold concentration was the first six amino acid residues of the putative rhGH prod-
achieved. Western blot analysis showed that (Figure 2), the mo- uct were Phe-Pro-Thr-Ile-Pro-Leu, identical to the true hGH
lecular mass of rhGH produced by B. subtilis BGSC-1A751 car- sequence. In support of this, nucleotide sequencing results
rying pMK4::pre(subC)::hGH and B. subtilis BGSC-1A197 were also 100% matching.
carrying pMK4::pre(subC)::hGH was 22 kDa being the same as
the standard hGH (Humatrope, Eli Lilly, France).
For further characterization, rhGH was purified, from 10-
fold concentrated and partially purified fermentation broth Host selection and effect of glucose concentration on
(Figure 3, Lane 1), by aptamer-based affinity chromatogra- rhGH fermentation
phy, which has been developed and is being studied in our Effects of initial glucose concentration on the recombinant
research group. Concentrated samples were mixed with hGH cells were investigated in laboratory-scale experiments by B.
specific aptamer and hGH-aptamer binding was carried out subtilis BGSC-1A751 and B. subtilis BGSC-1A197 carrying
at 25 C for 30 min (Figure 3, Lane 2). To obtain higher pu- pMK4::pre(subC)::hGH, at the initial concentrations of
rification, the aptamer-affinity separation step was applied CGo ¼ 6.0, 8.0, 10.0, and 15.0 g LÀ1. The variations in glu-
second time and after the elution step, rhGH was found to cose, cell and rhGH concentrations with the cultivation time
be separated from the fermentation broth with 99.8% purity by B. subtilis BGSC-1A751 carrying pMK4::pre(subC)::hGH
and 41% overall yield (Figure 3, Lane 3), the molecular are presented in Figures 5a–c, respectively. The cell concen-
mass of purified rhGH was determined by MALDI-ToF mass tration was not affected from the initial glucose concentra-
spectrometry (MS), to verify the structure of the secreted tion at t ¼ 0–6 h. Because of the addition of the protease
recombinant hormone. A commercial preparation (standard) inhibitor at t ¼ 6 h, an interruption in the cell growth was
of rhGH was analyzed first, and showed a spectral peak at observed until t ¼ 15 h. However, after t ¼ 15 h the second
m/z 22,126 (Figure 4a). The stated molecular mass of the cell growth phase was started with the initiation of rhGH
standard hormone is 22,125 Da. Thus, the detected ion was synthesis (Figure 5c). The highest cell concentration was
[MþH]þ, with the exact molecular mass of m ¼ 22,126 Da, obtained at CGo ¼ 15 g LÀ1 at t ¼ 36 h as CX ¼ 2.8 g LÀ1
where the charge on the ion is z ¼ þ1. The native length of (Figure 5a). In the first 15 h, the glucose consumption rates
the protein was obtained as the peak at m/z 22,133 (Figure were close to each other; however at t [ 15 h, parallel to
4b), corresponding to the [MþH]þ ion of rhGH detected the cell growth profiles, with the increase in cell growth rate,
with just a 0.03% error, which is a reasonable error at high the glucose consumption rate increased being the highest at
molecular weights in MALDI-ToF MS analysis. This result CGo ¼ 15 g LÀ1. The highest rhGH was produced at CGo ¼
indicates that rhGH molecule was synthesised by the 8 g LÀ1 at t ¼ 36 h as CrhGH ¼ 30 mg LÀ1. On the other
recombinant construct pMK4::pre(subC)::hGH, and then hand, when the protease inhibitor was not used rhGH was
secreted into the fermentation medium properly. not detected in the fermentation broths of B. subtilis BGSC-
The amino acid sequence of the signal peptide fused in 1A751 and B. subtilis BGSC-1A197 carrying pMK4::pre
front of the rhGH was: ‘‘MMRKKSFWLGMLTA (subC)::hGH.
FMLVFTMAFSDSASA;’’ and, the N-terminal and mass A parallel set of experiments were conducted by B. subti-
spectrometry analyses indicated that the SAP signal peptide lis BGSC-1A197 carrying pMK4::pre(subC)::hGH. Similar
was properly processed by the B. subtilis signal peptidase to B. subtilis BGSC-1A751 results, the highest rhGH produc-
(Spase I), because the results of N-terminal sequencing of tion was obtained at CGo ¼ 8 g LÀ1 but with a lower rhGH](https://image.slidesharecdn.com/hgh-101019014729-phpapp01/85/H-gh-power-resources-4-320.jpg)
H gh power resources
- 1. Expression System for Recombinant Human Growth Hormone Production from Bacillus subtilis ¸ ¨ ¨ ¨ ¨ Tuncer H. Ozdamar, Birgul S enturk, Ozge Deniz Yilmaz, and Guzide Calık ¨ ¸ Biochemical Reaction Engineering Laboratory, Chemical Engineering Dept., Ankara University, 06100 Ankara, Turkey Eda Celik and Pınar Calık ¸ ¸ Industrial Biotechnology and Metabolic Engineering Laboratory, Chemical Engineering Dept., Middle East Technical University, 06531 Ankara, Turkey DOI 10.1021/bp.81 Published online January 8, 2009 in Wiley InterScience (www.interscience.wiley.com). We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacil- lus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre-) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg-promoter in B. subtilis. Recombinant-hGH (rhGH) pro- duced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal-peptidase. The highest rhGH concentration was obtained at t ¼ 32 h as CrhGH ¼ 70 mg LÀ1 with a product yield on substrate YrhGH/S ¼ 9 g kgÀ1, in a glucose based defined medium. Fermentation charac- teristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic-acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino-acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for het- erologous protein production from Bacillus species. V 2009 American Institute of Chemical C Engineers Biotechnol. Prog., 25: 75–84, 2009 Keywords: Bacillus, recombinant, protein, human growth hormone, degQ, signal peptide, expression, secretion, MALDI-MS, fermentation Introduction and physiology of Gram-positive bacteria, and particularly of sporulation and associated metabolism4; whereupon, infor- Human growth hormone (hGH) is anionic, nonglycosylated mation concerning its secretion mechanism has been gath- four helix-bundle protein known as somatotropin, having a ered as the genome sequence was resolved.5 Nevertheless, molar mass of 22 kDa and 191 amino acid residues. It has the secretion of heterologous recombinant proteins from the been used to treat hypopituitary dwarfism, injuries, bone frac- bacilli might be inefficient. On the basis of the growing tures, bleeding ulcers, and burns.1 Recently, it appears to be of availability of information on genomics and proteomics of considerable benefit to girls with Turner’s syndrome, children B. subtilis, difficulties can now be systematically addressed with chronic renal failure, and adults with growth hormone and overcome.6 For the secretion of a recombinant protein deficiency or human immunodeficiency virus (HIV) syndrome.2 produced, either protease deficient Bacillus cells7 or protease Bacillus species, producers of several industrial enzymes, inhibitors are used. Westers et al.,8 in their review article, are potential hosts for production of heterologous nonglyco- summarised the efforts employed to improve B. subtilis as a sylated proteins of commercial interest. The advantages of host for protein secretion. Expression and secretion of non- using the Gram positive bacteria, besides the ability to glycosylated proteins in the genus Bacillus require the assis- secrete functional extracellular proteins directly into the bio- tance of the N-terminal signal-sequence of precursors. reactor culture medium, are the lack of pathogenicity and the Brockmeier et al.9 and Fu et al.10 reported the use of various absence of lipopolysaccharides (endotoxins) from the cell promoters and signal DNA sequences for recombinant pro- wall.3 Amongst, Bacillus subtilis has become a model system tein production by B. subtilis. for the study of many aspects of the biochemistry, genetics, Extracellular production of a recombinant foreign protein from a B. subtilis host requires a neat design and engineering Correspondence concerning this article should be addressed to T. H. of an expression and secretion system; wherein, the choice ¨ Ozdamar at [email protected]. of the promoter and signal DNA sequence in combination V 2009 American Institute of Chemical Engineers C 75
- 2. 76 Biotechnol. Prog., 2009, Vol. 25, No. 1 Table 1. Strains, Plasmids and Primers used in this Study Name Description Reference or Source Strain Bacillus licheniformis Wild type carrying subC gene DSM 1969 (11) B. subtilis nprÀaprÀ BGSC- 1A751 B. subtilis spoÀ BGSC- 1A179 Escherichia coli XL1Blue Plasmids pHGH107 (12) pUC19 (13) pMK4 (14) pUC19::pre(subC)::hGH This work pMK4::pre(subC)::hGH This work Primers for pre(subC)::hGH pre(subC) forward primer 50 _GCT CTA GAG CGC AAT CTC CTG TCA TTC G_30 Complimentary strand to hGH þ 50 _GGT ATA GTT GGG AAA GCA GAA GCG GAA TCG_30 pre(subC) reverse Complimentary strand to pre(subC) þ 50 _GCT TCT GCT TTC CCA ACT ATA CCA CTA TCT C_30 hGH forward primer hGH reverse primer 50 _GCG GAT CCG CAC TGG GGA GGG GTC AC_30 with the DNA vector, in particular, is important. For accession number A00501) from Homo sapiens and antibi- recombinant protein production using Bacillus species, there otic resistance genes to ampicillin and tetracycline. is no work in the literature reporting on the use of the pro- The primers used for the amplification are given in Table moter and signal sequence of the DNA encoding the indus- 1. Signal DNA sequence of subC was fused in front of the trial enzyme serine alkaline protease (SAP). hGH gene using gene splicing by overlap extension The idea in this work is based on the construction of a method.20 XbaI restriction site was incorporated to the for- recombinant plasmid for the synthesis and secretion of rhGH ward primer of pre(subC) sequence, whereas BamHI restric- that mimics the synthesis of SAP in bacilli. Thus, the tion site was incorporated to the reverse primer of hGH hybrid-gene of two DNA fragments, i.e., signal (pre-) DNA gene. To verify the cloning, nucleotide sequencing analyses sequence of B. licheniformis serine alkaline protease (SAP) were performed at Microsynth GmbH (Switzerland) using gene (subC) and cDNA encoding hGH, were cloned into the designed primers. pMK4 plasmid and expressed under the deg-promoter in a B. subtilis host. Production of rhGH from B. subtilis and the fermentation characteristics in a defined medium were inves- Culture maintenance and media for fermentation tigated, using the designed hybrid-gene system. For the bioprocess experiments, B. subtilis BGSC-1A751 (nprÀaprÀ) and B. subtilis BGSC-1A197 (spoÀ) stock cul- Experimental Methods tures were maintained on agar slants that contained (g LÀ1): peptone, 5; beef extract, 3; agar, 15; and initial pH ¼ 7.25. Bacterial strains, plasmids, and growth media for The cells on the newly prepared slants were inoculated into genetic manipulation the preculture medium for preparation of inocula that con- The strains, plasmids, and primers used in this study are tained (g LÀ1): soytryptone, 15; peptone, 5; MnSO4.2H2O, described in Table 1. Bacterial strains, plasmids, and growth 0.010; Na2HPO4, 0.25; CaCl2, 0.100 and grown at 37 C for media were prepared using standard techniques.15 B. licheni- 6 h. The defined reference production medium for batch-bio- formis (DSM 1969), B. subtilis, and Escherichia coli XL1- reactor was as follows (g LÀ1): glucose, 6.0; (NH4)2HPO4, Blue16 were maintained and grown on LB-agar that con- 4.7; KH2PO4, 2.0; 0.04 M Na2HPO4 and NaH2PO4; the ini- tained (g LÀ1): tryptone, 10; NaCl, 5; yeast extract, 5; agar, tial pH ¼ 7.25.11,21 Chloramphenicol (7 lg/mL) was used in 15 and in LB broth (without agar) at 37 C. Ampicillin (100 all bioprocess experiments of plasmid-bearing B. subtilis. lg/mL) was used for the plasmid maintenance in E. coli Complete EDTA-free protease inhibitor (Roche) was used to strains; 7 lg/mL chloramphenicol was used for plasmid prevent proteolytic hydrolysis of the produced rhGH. maintenance in the recombinant B. subtilis. Laboratory-scale batch fermentations Manipulation of DNA, PCR, cloning, and DNA sequencing Batch laboratory-scale fermentation experiments were con- B. licheniformis chromosomal DNA was isolated as ducted in orbital shakers under agitation and heating rate described by Posprech and Neumann.17 subC gene (GenBank control, using air-filtered 500 mL Erlenmeyer-flasks having Acc. No. X03341)18,19 that encodes for extracellular serine 220 mL working volume capacities. Batch-bioreactor experi- alkaline protease (SAP) enzyme of B. licheniformis was used ments were conducted in 1.0 L bioreactor systems (BBraun, as the template for amplification of signal (pre-) sequence. Germany) consisted of temperature, pH, foam, air inlet, and HGH cDNA16 was amplified from E. coli host strain carry- stirring rate controls with 0.5 L working volume. Each ing the plasmid pHGH107 (ATCC 31538; US patent no. experiment was conducted in two bioreactors operating in 4,342,832), featuring the growth hormone ORF (NCBI parallel, to check reproducibility.
- 3. Biotechnol. Prog., 2009, Vol. 25, No. 1 77 Analyses microliters of a 10 mg mLÀ1 sinapinic acid matrix dissolved Cell concentrations based on dry weights were measured in 50% acetonitrile and 0.1% TFA solution, was mixed with with a UV-vis spectrophotometer (Shimadzu UV-160A, To- 1 lL of $10 pmol lLÀ1 sample and 1 lL of this mixture kyo, Japan) using a calibration curve obtained at 600 nm. was spotted on the target plate and air-dried by the ‘‘dried Glucose consumption was followed by the glucose oxidation droplet’’ technique.28 Cytochrome c and Humatrope (stand- method at 505 nm with UV-vis spectrophotometer.22 ard hGH) were used as molecular weight standards for pur- Excreted amino acid concentrations were measured with an poses of mass correction. Spectra were generated from the amino acid analysis system (Waters HPLC, Milford, MA), sum of 100–200 laser pulses and mass determinations were using the Pico Tag method.23 Excreted organic acid concen- made by finding the peak centroid of a smoothed signal (by trations were measured with an HPLC (Waters, HPLC, Alli- Savitzky-Golay algorithm) after background subtraction.29 ance 2695).24 HGH concentrations were measured using a high-performance capillary electrophoresis (Waters HPCE, Results Quanta 4000 E, Milford, MA). The samples were analyzed at 12 kV and 15 C with a positive power supply using 60 cm Construction of the plasmid pMK4::pre(subC)::hGH Â 75 lm silica capillary using modified 100 mM borate B.licheniformis (DSM 1969) chromosomal DNA and buffer (pH ¼ 10) including zwitterions (Z1-Methyl, Waters) pHGH107 plasmid, containing the hGH cDNA, were isolated as the separation buffer. Proteins were detected by UV ab- to be used as templates in PCR reactions. The two target sorbance at 214 nm, as mentioned elsewhere.23,25 Humatrope genes, pre(subC) of subC gene (360 bp) from B. lichenifor- (Eli Lilly, France) was used as the standard. The Dynamic mis chromosomal DNA and mature peptide sequence of method26 was applied to find the oxygen uptake rate (OUR) hGH (639 bp) from pHGH107 plasmid, were amplified by and oxygen transfer coefficient (KLa) values. PCRs (Figure 1). The primers (Table 1) used at the ends to The physiological data for each operation were from at be joined were designed as complementary to one another least two independent experiments, and the average values by including nucleotides at their 50 ends that are complemen- were given. tary to the 30 portion of the other primer. The two PCR prod- ucts containing the overlapping fragments at the ends to be Ultrafiltration and purification joined were purified and, by the third PCR reaction using the Concentration and desalting of the production medium external primers carrying XbaI and BamHI restriction sites, was achieved by ultrafiltration under nitrogen gas (55 psi, extension of the overlap by DNA polymerase has yielded the 3.8 bar) at 4 C using Amicon 400 mL stirred pressure cells hybrid-gene product, i.e., pre(subC)::hGH (999 bp), where (Millipore, Bedford, MA) with regenerated cellulose ultrafil- pre(subC) DNA sequence, was fused in front of the hGH tration membranes having MWCO of 10 kDa (Millipore, sequence (Figure 1). The hybrid-gene pre(subC)::hGH was Bedford, MA). Purification of rhGH was achieved by then cloned into the XbaI and BamHI sites of pUC19 E. coli aptamer-based affinity chromatography. Concentrated sam- plasmid, and transformed into E. coli XLI-Blue cells by ples were mixed with hGH specific aptamer which was im- CaCl2 method. Thereafter, pre(subC)::hGH was sub-cloned mobilized onto microparticles and hGH-aptamer binding was to pMK4 SalI and BamHI sites and expressed in the hosts B. carried out at 25 C for 30 min, which has been developed subtilis BGSC-1A751 (nprÀaprÀ) and B. subtilis BGSC- and is being studied in our research group. 1A197 (spoÀ). SDS-PAGE, Western Blotting, and N-terminal SDS-PAGE, Western Blot, N-terminal, and mass sequence analysis spectrometry analyses Sodium dodecyl sulfate-polyacrylamide gel electrophoresis RhGH production potential of the recombinant cells, B. subti- (SDS-PAGE) was performed as described by Laemmli27 by lis BGSC-1A751 (nprÀaprÀ) carrying pMK4::pre(subC)::hGH using 4% stacking and 12% separating polyacrylamide gel, run on a Mini Protean II DUAL SLAB cell (Bio-Rad) according to the manufacturer’s instructions and silver stained. For West- ern blot analysis, polyclonal rabbit anti-human growth hor- mone (BioMeda, USA) was used as the primary antibody and horseradish peroxidase labeled goat-anti rabbit IgG (HþL) (BioMeda) was used as the secondary antibody. For the N-ter- minal analysis, rhGH was electrophoresed as described above and transferred onto a polyvinylidene difluoride membrane (Millipore, USA). After being stained with Coomassie blue, the rhGH band was excised, and automated Edman degrada- tion was performed by PROCISE 494 gas-phase/liquid-pulse sequencer (Applied Biosystems, Foster City, CA). MALDI-ToF mass spectrometry analysis The molecular weight of rhGH was determined by the use of a MALDI-LR (Waters-Micromass, UK) instrument. Spec- Figure 1. Agarose gel electrophoresis view for PCR amplifica- tra were generated using a pulsed nitrogen gas laser tion of hGH, pre(subC), and pre(subC)::hGH. (337 nm) in positive linear mode with a low mass gate of M, low range marker (Fermantas); Lane 1, hGH; Lane 2, pre 1,000 Da.25 The accelerating voltage was 15 kV. Three (subC)::hGH; and Lane 3, pre(subC).
- 4. 78 Biotechnol. Prog., 2009, Vol. 25, No. 1 Figure 2. Western Blott analysis results of BGSC-1A751 (npr2, apr2) carrying pre(subC)::hGH and BGSC- 1A197 (spo2) carrying pre(subC)::hGH. Lane 1, commercial (standard) hGH; Lane 2, hGH produced by r-B. ‘subtilis BGSC-1A751 (nprÀ, aprÀ) carrying pMK4::pre (subC)::hGH; Lane 3, hGH produced by r-B. subtilis BGSC- 1A197 (spoÀ) carrying pMK4::pre(subC)::hGH; and Lane 4, marker (Sigma M 0671). Figure 3. SDS-PAGE analysis of rhGH, produced by r-B.subti- lis BGSC-1A751 (npr2, apr2) carrying pMK4:: pre(subC)::hGH. M, protein marker (Fermentas); Lane 1, product mixture of and B. subtilis BGSC-1A197 (spoÀ) carrying pMK4::pre r-B.subtilis containing rhGH; Lane 2, 1st rhGH separation with hGH specific aptamer; Lane 3, 2nd rhGH separation with hGH (subC)::hGH were determined on a glucose (CG ¼ 6 g LÀ1) specific aptamer; and Lane 4, standard hGH. based defined medium. The supernatant obtained by centrifuga- tion at t ¼ 27 h of the fermentation was partially purified by dead-end ultrafiltration and nearly 10-fold concentration was the first six amino acid residues of the putative rhGH prod- achieved. Western blot analysis showed that (Figure 2), the mo- uct were Phe-Pro-Thr-Ile-Pro-Leu, identical to the true hGH lecular mass of rhGH produced by B. subtilis BGSC-1A751 car- sequence. In support of this, nucleotide sequencing results rying pMK4::pre(subC)::hGH and B. subtilis BGSC-1A197 were also 100% matching. carrying pMK4::pre(subC)::hGH was 22 kDa being the same as the standard hGH (Humatrope, Eli Lilly, France). For further characterization, rhGH was purified, from 10- fold concentrated and partially purified fermentation broth Host selection and effect of glucose concentration on (Figure 3, Lane 1), by aptamer-based affinity chromatogra- rhGH fermentation phy, which has been developed and is being studied in our Effects of initial glucose concentration on the recombinant research group. Concentrated samples were mixed with hGH cells were investigated in laboratory-scale experiments by B. specific aptamer and hGH-aptamer binding was carried out subtilis BGSC-1A751 and B. subtilis BGSC-1A197 carrying at 25 C for 30 min (Figure 3, Lane 2). To obtain higher pu- pMK4::pre(subC)::hGH, at the initial concentrations of rification, the aptamer-affinity separation step was applied CGo ¼ 6.0, 8.0, 10.0, and 15.0 g LÀ1. The variations in glu- second time and after the elution step, rhGH was found to cose, cell and rhGH concentrations with the cultivation time be separated from the fermentation broth with 99.8% purity by B. subtilis BGSC-1A751 carrying pMK4::pre(subC)::hGH and 41% overall yield (Figure 3, Lane 3), the molecular are presented in Figures 5a–c, respectively. The cell concen- mass of purified rhGH was determined by MALDI-ToF mass tration was not affected from the initial glucose concentra- spectrometry (MS), to verify the structure of the secreted tion at t ¼ 0–6 h. Because of the addition of the protease recombinant hormone. A commercial preparation (standard) inhibitor at t ¼ 6 h, an interruption in the cell growth was of rhGH was analyzed first, and showed a spectral peak at observed until t ¼ 15 h. However, after t ¼ 15 h the second m/z 22,126 (Figure 4a). The stated molecular mass of the cell growth phase was started with the initiation of rhGH standard hormone is 22,125 Da. Thus, the detected ion was synthesis (Figure 5c). The highest cell concentration was [MþH]þ, with the exact molecular mass of m ¼ 22,126 Da, obtained at CGo ¼ 15 g LÀ1 at t ¼ 36 h as CX ¼ 2.8 g LÀ1 where the charge on the ion is z ¼ þ1. The native length of (Figure 5a). In the first 15 h, the glucose consumption rates the protein was obtained as the peak at m/z 22,133 (Figure were close to each other; however at t [ 15 h, parallel to 4b), corresponding to the [MþH]þ ion of rhGH detected the cell growth profiles, with the increase in cell growth rate, with just a 0.03% error, which is a reasonable error at high the glucose consumption rate increased being the highest at molecular weights in MALDI-ToF MS analysis. This result CGo ¼ 15 g LÀ1. The highest rhGH was produced at CGo ¼ indicates that rhGH molecule was synthesised by the 8 g LÀ1 at t ¼ 36 h as CrhGH ¼ 30 mg LÀ1. On the other recombinant construct pMK4::pre(subC)::hGH, and then hand, when the protease inhibitor was not used rhGH was secreted into the fermentation medium properly. not detected in the fermentation broths of B. subtilis BGSC- The amino acid sequence of the signal peptide fused in 1A751 and B. subtilis BGSC-1A197 carrying pMK4::pre front of the rhGH was: ‘‘MMRKKSFWLGMLTA (subC)::hGH. FMLVFTMAFSDSASA;’’ and, the N-terminal and mass A parallel set of experiments were conducted by B. subti- spectrometry analyses indicated that the SAP signal peptide lis BGSC-1A197 carrying pMK4::pre(subC)::hGH. Similar was properly processed by the B. subtilis signal peptidase to B. subtilis BGSC-1A751 results, the highest rhGH produc- (Spase I), because the results of N-terminal sequencing of tion was obtained at CGo ¼ 8 g LÀ1 but with a lower rhGH
- 5. Biotechnol. Prog., 2009, Vol. 25, No. 1 79 Figure 4. MALDI-ToF MS analysis. (a) Standard hGH (b) B. subtilis produced and purified rhGH. value (CrhGH ¼ 26 mg LÀ1). On the basis of the results, B. by B. subtilis carrying merely pMK4, as can be seen in Fig- subtilis BGSC-1A751 was selected as the host. ure 6. In the process by B. subtilis carrying pMK4, the glucose consumption was higher until t ¼ 18 h of the fermentation, but after t ¼ 18 h, it was almost zero; where the highest cell Influence of hGH gene on the physiology of r-Bacillus concentration and the overall cell yield on substrate (YX/S) subtilis were obtained as CX ¼ 1.6 g LÀ1 (t ¼ 12 h) and YX/S ¼ To determine the influence of hGH gene on the physiol- 0.23 g gÀ1, respectively. Contrarily, by B. subtilis carrying ogy of the bacilli, bioreactor experiments were performed by pMK4::pre(subC)::hGH, the glucose consumption was B. subtilis BGSC-1A751 carrying merely pMK4, and B. sub- increased after t ¼ 18 h and the highest cell concentration tilis BGSC-1A751 carrying pMK4::pre(subC)::hGH, at T ¼ was obtained as CX ¼ 2.0 g LÀ1 (t ¼ 24 h); and the overall 37 C, pH0 ¼ 7.25, CGo ¼ 8 g LÀ1, air inlet rate of 0.5 vvm cell yield on substrate was YX/S ¼ 0.25 g gÀ1. and agitation rate of 800 minÀ1. The concentrations of the As expected, rhGH production was achieved only by B. glucose, cell, extracellular rhGH, the by-products amino and subtilis carrying pMK4::pre(subC)::hGH. RhGH synthesis organic acids, together with the oxygen-uptake (OUR) rates, started at t ¼ 18 h of the batch-bioprocess and increased oxygen-transfer coefficients (KLa), and yield coefficients with the cultivation time reaching the value CrhGH ¼ 70 mg were determined throughout the fermentations. LÀ1 at t ¼ 32 h. The highest product yield on substrate was The variations in dissolved oxygen concentration (CO) and obtained at 24 t 32 h as YrhGH/S ¼ 0.65 g gÀ1, while pH with the cultivation time are presented in Figure 6; and the the overall rhGH yield on substrate was YrhGH/S ¼ 9 g kgÀ1. variations in glucose, cell, and rhGH concentrations are pre- The excreted amino acids that were detected at considerable sented in Figure 7. The loci of the CO vs. t profiles obtained concentrations in both fermentations are leucine, isoleucine, in the two fermentation processes were similar until t ¼ 18 h, and phenylalanine. The highest concentrations of leucine, iso- where considerable decrease in dissolved oxygen concentration leucine, and phenylalanine by B. subtilis carrying pMK4::pre was observed at t ¼ 0–4 h. However, in the process by B. sub- (subC)::hGH were 0.191, 0.096, and 0.132 g LÀ1; whereas by tilis carrying pMK4::pre(subC)::hGH, a considerable decrease B. subtilis carrying pMK4 were as 0.214, 0.107, and 0.281 g in CO between t ¼ 18 h and t ¼ 22 h was detected due to LÀ1, respectively. Nevertheless, alanine, arginine, asparagine, rhGH synthesis that induced the cell growth. By the termina- aspartic acid, glutamic acid, glycine, histidine, methionine, ly- tion of the cell formation, a breakthrough in dissolved oxygen sine, valine, treonine, and tryptophan were not detected in the concentration at t ¼ 22 h is observed. broths of the two fermentation processes. Thus, the total The pH in both fermentation media decreased until t ¼ excreted amino acid concentrations were higher in the fermen- 18 h. After t ¼ 18h, pH continued to decrease by depicting tation by B. subtilis carrying merely pMK4. a characteristic curve by B. subtilis carrying pMK4::pre Variations in excreted organic acid concentration produced (subC)::hGH; contrarily, pH was increased in the bioreactor by B. subtilis carrying pMK4::pre(subC)::hGH and B.
- 6. 80 Biotechnol. Prog., 2009, Vol. 25, No. 1 Figure 5. (a) Variation in cell concentration with the cultivation time for B. subtilis BGSC-1A751 (npr2apr2) carrying pMK4::pre (subC)::hGH with the initial glucose concentration. CGo(g L21): (^) 6.0; () 8.0; (~) 10.0; (*) 15.0. (b) Variation in glu- cose concentration with the cultivation time for B. subtilis BGSC-1A751 (npr2apr2) carrying pMK4::pre(subC)::hGH with the initial glucose concentration. CGo(g L21): (^) 6.0; () 8.0; (~) 10.0; (*) 15.0. (c) Variation in rhGH concentration with the cultivation time for B. subtilis BGSC-1A751 (npr2apr2) carrying pMK4::pre(subC)::hGH with the initial glucose concentration. CGo(g L21): (^) 6.0; () 8.0; (~) 10.0; (*) 15.0 as in 5c. subtilis carrying pMK4 are presented in Figures 8a,b, respec- Fermentation characteristics tively. Oxaloacetic acid, which is known to be produced in The variations in KLa and the oxygen uptake rate (OUR) cell regeneration, was not excreted in both fermentations. In are presented in Table 2. Considering the characteristic cell the fermentation by B. subtilis carrying pMK4::pre and rhGH concentration profiles of B. subtilis carrying (subC)::hGH, the main extracellular by-products were suc- pMK4::pre(subC)::hGH, the bioprocess was divided into five cinic, gluconic and formic acid (Figure 8a). Lactic, oxalic, periods. 0 t 4 h is the cell first-growth-phase; 4 t citric acids are the organic acids having lower concentra- 12 h is the growth-interruption-phase; 12 t 16 h is the tions, i.e., ca. 0.1 g LÀ1; moreover, pyruvic, a-ketoglutaric, and acetic acids were detected at a concentration of ca. lag-phase where rhGH synthesis starts; 16 t 24 h is the 0.01 g LÀ1. Indeed, the organic acid profiles obtained by B. second-cell-growth-phase where rhGH synthesis increases; subtilis carrying pMK4::pre(subC)::hGH are different than and 24 t 32 h is the end of the growth-phase where that of B. subtilis carrying pMK4; where in the latter, the rhGH synthesis was the highest. main excreted by-products were malic and gluconic acids In both fermentation processes, the oxygen transfer coeffi- (Figure 8b), and the concentration of the other organic acids cient, KLa, increased with the increase in the cultivation were lower than 0.01 g LÀ1. Thus, the amount of total or- time, and then decreased. At 0 t 4 h of the bioprocess, ganic acids excreted by B. subtilis carrying pMK4::pre KLa and oxygen uptake rate of B. subtilis carrying pMK4 (subC)::hGH were higher. was higher than that of B. subtilis carrying hGH gene.
- 7. Biotechnol. Prog., 2009, Vol. 25, No. 1 81 Figure 6. Variations in dissolved oxygen concentration and pH with the cultivation time by B.subtilis BGSC-1A751 Figure 7. Variations in glucose, cell, and hGH concentrations (npr2apr2) carrying pMK4::pre(subC)::hGH (r- with the cultivation time by B. subtilis BGSC-1A751 pMK4) and BGSC-1A751 (npr2apr2) carrying (npr2apr2) carrying pMK4::pre(subC)::hGH and pMK4. Co: continuous lines, pH: dashed lines. BGSC-1A751 (npr2apr2) carrying pMK4. Glucose concentration: (--n--) pMK4; () pMK4::pre (subC)::hGH, Cell concentration: (--l--) pMK4; (*) Throughout the bioprocess, the highest KLa value was pMK4::pre(subC)::hGH, rhGH concentration: (~) obtained by B. subtilis carrying merely pMK4 as KLa¼ pMK4::pre(subC)::hGH. 0.028 sÀ1 at 0 t 4 h; however, due to low oxygen con- centrations within t ¼ 4–16 h in the production medium, the dynamic method could not be applied. Related with B. subti- spectrometry analyses indicate that the signal peptidase has lis carrying pMK4::pre(subC)::hGH, the highest OUR values cut at the site within Spase I group. Thus, the system were obtained in the first growth- (0 t 4 h) and second- designed functioned with its intended purpose effectively in cell-growth- phases (16 t 24 h) (Table 2). expression and cleavage of the recombinant product. The other peak in Figure 4b at m/z 25,854 is possibly an unspecifically bound impurity protein to the hGH-ligand during Discussion and Conclusions purification. From the facts that MALDI-ToF MS can not be used in quantitation of proteins because protein detection An expression system producing therapeutic protein depends on ionisation efficiency and that even femto-mole human growth hormone that conceptually mimics the extrac- amounts can be detected, and from SDS-PAGE (Figure 3, ellular serine alkaline protease synthesis in the genus Bacil- Lane 3) analysis where a 25.8 kDa band was not detected, it lus was designed and implemented. For the extracellular was concluded that the impurity was in negligible amounts. production of human growth hormone by B. subtilis, a Furthermore, the peaks detected at m/z 20.4 kDa in Figures recombinant plasmid carrying the hybrid-gene of two DNA 4a,b, are possibly the cleaved 20 kDa forms of hGH.32 fragments, i.e., signal (pre-) DNA sequence of a Bacillus The constructed expression system produces extracellular licheniformis extracellular SAP enzyme gene (subC) and the rhGH from B. subtilis starting from the beginning of the fer- DNA encoding hGH, was constructed and transferred into mentation process parallel to the cell growth, giving a break- two host Bacillus strains, namely B. subtilis BGSC-1A751 through at t ¼ 12 h. RhGH concentration was the highest at (nprÀaprÀ) and B. subtilis BGSC-1A197 (spoÀ). These t ¼ 32 h as CrhGH ¼ 70 mg LÀ1 and overall specific-product strains were selected for their deficiencies in two protease yield on substrate was YrhGH/S ¼ 9 g kgÀ1, in the defined genes and sporulation gene, respectively. RhGH, expressed medium with sole carbon source glucose. Nakayama et al.,33 by the hybrid-gene pre(subC)::hGH cloned into pMK4 in reported rhGH secretion level of 40 mg LÀ1 which is 1.75- both hosts, was secreted. fold lower than that obtained in this study. Kajino et al.,34 The approach developed is expected to be applicable to modified the ‘‘middle wall protein (MWP) signal peptide’’ of the design of expression systems for heterologous protein B. brevis and constructed a hGH expression system and productions from Bacillus. As the rhGH concentration reported rhGH secretion from B. brevis with an overall spe- obtained from the protease-deficient host B. subtilis BGSC- cific-product yield on glucoseþpolypeptone YrhGH/S ¼ 4 g 1A751 was higher than that of the host B. subtilis BGSC- kgÀ1 which is 2.25-fold lower than the value reported in this 1A197, the first was selected as the host owing to two gene work. Related with a different expression system for human deletions targeting the decrease in protease activities. interleukin-3, Westers et al.,35 reported 0.1 g LÀ1 recombi- Secreted proteins are generally synthesised as precursors nant human interleukin-3 secretion by an eight-protease-defi- with a cleavable signal peptide, and then the signal peptide cient B. subtilis, using a semi-defined enriched medium; is removed by signal peptidases, where preprotein processing where the overall specific-product yield on substrate was by signal peptidases are essential for bacterial growth and vi- lower then the YrhGH/S value reported in this work. There- ability.30,31 The native length of rhGH was detected in SDS- fore, we conclude that the expression system designed, PAGE (Figure 2, Lane 2), Western blot (Figure 3, Lane 3) which is based on the idea of using the ribosomal binding and MALDI-ToF MS analysis as the peak at m/z 22,133 site- promoter- and the signal peptide of serine alkaline pro- (Figure 4b) with just a 0.03% error, which is a reasonable tease enzyme gene subC, is successful for the extracellular error at high molecular weights. The N-terminal and mass recombinant protein production.
- 8. 82 Biotechnol. Prog., 2009, Vol. 25, No. 1 Figure 8. (a) Variation in organic acid concentrations with the cultivation time by B. subtilis BGSC-1A751 (npr2apr2) carrying pMK4::pre(subC)::hGH. (b) Variation in organic acid concentrations with the cultivation time by B.subtilis BGSC-1A751 (npr2apr2) carrying pMK4. Table 2. Variation in Oxygen Transfer Characteristics with the Cultivation Time OUR*103 Microorganism Period KLa (sÀ1) (mol mÀ3 sÀ1) BGSC-1A751 (nprÀaprÀ) First growth phase, 0 t 4 h 0.017 3.5 carrying pMK4::pre(subC)::hGH Growth-interruption-phase, 4 t 12 h 0.018 3.0 Lag-phase and rhGH 0.014 2.4 synthesis phase, 12 t 16 h Second cell growth and rhGH 0.015 3.5 synthesis phase, 16 t 24 h End of the growth and rhGH 0.010 0.4 synthesis phase, 24 t 32 h À À BGSC-1A751 (npr apr ) 0t4 h 0.028 4.6 carrying pMK4 4 t 12 h – – 12 t 16 h – – 16 t 24 h 0.013 0.4 24 t 32 h 0.010 0.3 The transcription for rhGH synthesis by B. subtilis BGSC- (subC)::hGH, on glucose as sole carbon source. The results 1A751 (nprÀaprÀ) carrying the hybrid-gene pre reveal that the expression of rhGH influences the physiology (subC)::hGH is under the control of degQ promoter; there- of the r-Bacillus cells, as expected. The cell concentration fore, the synthesis and secretion pattern of rhGH mimics the profile of B. subtilis carrying pMK4::pre(subC)::hGH shows synthesis and secretion of SAP enzyme in B. lichenifor- a perturbed biphasic variation because of the introduction of mis.11,21,36 Because the ribosomal binding site, promoter, a protease inhibitor at t ¼ 4 h, where a drastic decrease in and signal peptide of the SAP gene (subC) were used in the the growth rate occurs until t ¼ 16 h, that proceeds with an constructed expression system for the synthesis and secretion increase in the growth rate until ca. t ¼ 24 h. of rhGH, a similar rhGH concentration profile to that of RhGH synthesis and secretion proceeded until t ¼ 32 h, the SAP productions from B. licheniformis carrying giving the highest concentration as CrhGH ¼ 70 mg LÀ1. As pHV1431::subC21 and B. subtilis carrying pHV1431::subC11 expected, there was no rhGH production by the microorgan- was obtained. The slight difference observed in the concen- ism that does not carry hGH gene. tration profiles was likely due to an interruption caused by Due to the introduction of new biochemical reactions into the addition of protease inhibitors. Therefore, we conclude the intracellular reaction network producing the heterologous that the expression and secretion system constructed for extracellular protein, the fermentation and oxygen transfer rhGH production from the genus Bacillus is dependent on characteristics and by-product distributions of B. subtilis car- the bioreactor operation conditions (which is being studied), rying pMK4::pre(subC)::hGH were different than that of the similar to that of SAP production by Bacillus species.37 B. subtilis carrying merely pMK4. Moreover, higher concen- To investigate the influence and perturbation effect of trations of organic acids detected in the broth of B. subtilis hGH gene on the physiology of r-B. subtilis, comparative carrying pMK4::pre(subC)::hGH, and higher concentrations bioreactor experiments were performed by B. subtilis carry- of amino acids detected in the broth of B. subtilis carrying ing merely pMK4 and B. subtilis carrying pMK4::pre pMK4, reveal the impact of the structure of the plasmids on
- 9. Biotechnol. Prog., 2009, Vol. 25, No. 1 83 the synthesis capacity of the host throughout the Moestl D, Nakai S, Noback M, Noone D, OReilly M, Ogawa fermentation. K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Por- tetelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, The expression system carrying the foreign gene, hGH, in Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Riv- the hybrid-gene fused behind the signal (pre-) DNA olta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scan- sequence, i.e., pre(subC), synthesizing and secreting rhGH lan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, from B. subtilis carrying pMK4::pre(subC)::hGH, was influ- Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin enced by the bioreactor operation conditions which, in turn A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi effects the oxygen transfer characteristics of the fermentation M, Tamakoshi A, Tanaka T, Terpstra P, Tognoni A, Tosato V, Uchiyama S, Vandenbol M, Vannier F, Vassarotti A, Viari process, in the defined medium based on sole carbon source A, Wambutt R, Wedler E, Wedler H, Weitzenegger T, Win- glucose. 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Boland F, Brignell SC, Bron S, Bunai K, Chapui J, Christiansen The bioreactor operation conditions applied for the LC, Danchin A, Debarbouille M, Dervyn E, Deuerling E, fermentation experiments were the favourable conditions Devine K, Devine SK, Dreesen O, Errington J, Fillinger S, Fos- that were found for the SAP production by B. licheniformis ter SJ, Fujita Y, Galizzi A, Gardan R, Eschevins C, Fukushima T, Haga K, Harwood CR, Hecker M, Hosoya D, Hullo MF, carrying pHV1431::subC21 and B. subtilis carrying Kakeshita H, Karamata D, Kasahara Y, Kawamura F, Koga K, pHV1431::subC.11 The fermentation and oxygen-transfer Koski P, Kuwana R, Imamura D, Ishimaru M, Ishikawa S, Ishio characteristics reveal that the extracellular rhGH production I, Le Coq D, Masson A, Mauel C, Meima R, Mellado RP, Moir proceeds through the constructed expression system by form- A, Moriya S, Nagakawa E, Nanamiya H, Nakai S, Nygaard P, ing unique intracellular reaction pathways with different in- Ogura M, Ohanan T, O’Reilly M, O’Rourke M, Pragai Z, Poo- tracellular reaction rates compared to that of SAP ley HM, Rapoport G, Rawlins JP, Rivas LA, Rivolta C, Sadaie production. 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