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High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum

  • Applied genetics and molecular biotechnology
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Abstract

We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in Corynebacterium glutamicum. For efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5′ untranslated region (5′ UTR) sequence, and (5) transcriptional terminator. Among all the systems examined, the use of a codon-optimized gene sequence, a Sec-dependent PorB signal peptide, and a fully synthetic H36 promoter, allowed the highest production of antibody fragments in a culture medium. For large-scale production, fed-batch cultivations were also conducted in a 5-L lab-scale bioreactor. When cells were cultivated in semi-defined media, cells could grow up to an OD600 of 179 for 32 h and an antibody fragment concentration as high as 68 mg/L could be obtained in a culture medium with high purity. From the culture medium, the secreted antibody was successfully purified using a simple purification procedure, with correct binding activity confirmed by enzyme-linked immunosorbent assay. To the best of our knowledge, this is the first report of a fed-batch cultivation for antibody fragment production in C. glutamicum.

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References

  • Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M (2012) scFv antibody: principles and clinical application. Clin Dev Immunol 2012:980250

  • Aldor IS, Krawitz DC, Forrest W, Chen C, Nishihara JC, Joly JC, Champion KM (2005) Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical. Appl Environ Microbiol 71(4):1717–1728

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • An SJ, Yim SS, Jeong KJ (2013) Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide. Protein Expr Purif 89:251–257

    Article  CAS  PubMed  Google Scholar 

  • Buss NA, Henderson SJ, Mcfarlane M, Shenton JM, de Haan L (2012) Monoclonal antibody therapeutics: history and future. Curr Opin Pharmacol 12:615–622

    Article  CAS  PubMed  Google Scholar 

  • Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30:1102–1107

    Article  CAS  PubMed  Google Scholar 

  • Dammeyer T, Steinwand M, Krüger SC, Dübel S, Hust M, Timmis KN (2011) Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory. Microb Cell Fact 10:11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Date M, Itaya H, Matsui H, Kikuchi Y (2006) Secretion of human epidermal growth factor by Corynebacterium glutamicum. Lett Appl Microbiol 42(1):66–70

    Article  CAS  PubMed  Google Scholar 

  • Eikmanns BJ (1992) Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and thiosephosphate isomerase. J Bacteriol 174(19):6076–6086

    CAS  PubMed Central  PubMed  Google Scholar 

  • Ermolaeva MD (2001) Synonymous codon usage in bacteria. Curr Issues Mol Biol 3(4):91–97

    CAS  PubMed  Google Scholar 

  • Harvey BR, Georgiou G, Hayhurst A, Jeong KJ, Iverson BL, Rogers GK (2004) Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc Natl Acad Sci U S A 101:9193–9198

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hong HJ, Kim ST (2002) Antibody engineering. Biotechnol Bioprocess Eng 7:150–154

    Article  CAS  Google Scholar 

  • Huang CJ, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39(3):383–399

    Article  CAS  PubMed  Google Scholar 

  • Jang SH, Jeong KJ (2013) Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli. Protein Expr Purif 89(2):169–174

    Article  CAS  PubMed  Google Scholar 

  • Jeong KJ, Jang SH, Velmurugan N (2011) Recombinant antibodies: engineering and production in yeast and bacterial hosts. Biotechnol J 6(1):16–27

    Article  CAS  PubMed  Google Scholar 

  • Jiang L, He L, Fountoulakis M (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J Chromatogr A 1023:317–320

    Article  CAS  PubMed  Google Scholar 

  • Jo SJ, Matsumoto K, Leong CR, Ooi T, Taguchi S (2007) Improvement of poly(3-hydroxybutyrate) [P(3HB)] production in Corynebacterium glutamicum by codon optimization, point mutation and gene dosage of P(3HP) biosynthetic genes. J Biosci Bioeng 104(6):457–463

    Article  CAS  PubMed  Google Scholar 

  • Katsuda T, Sonoda H, Kumada Y, Yamaji H (2012) Production of antibody fragments in Escherichia coli. Methods Mol Biol 907:305–324

    Article  CAS  PubMed  Google Scholar 

  • Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H (2003) Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces albogriseolus. Appl Environ Microbiol 69(1):358–366

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kikuchi Y, Itaya H, Date M, Matsui K, Wu LF (2009) TatABC overexpression improves Corynebacterium glutamicum Tat-dependent protein secretion. Appl Environ Microbiol 75:603–607

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kind S, Jeong WK, Schröder H, Wittmann C (2010) Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane. Metab Eng 12(4):341–351

    Article  CAS  PubMed  Google Scholar 

  • Lee YJ, Jeong KJ (2013) Enhanced production of antibody fragment via SRP pathway engineering in Escherichia coli. Biotechnol Bioprocess Eng 18:751–758

    Article  CAS  Google Scholar 

  • Lee YJ, Kim HS, Ryu AJ, Jeong KJ (2013) Enhanced production of full-length immunoglobulin G via the signal recognition particle (SRP)-dependent pathway in Escherichia coli. J Biotechnol 165:102–108

    Article  CAS  PubMed  Google Scholar 

  • Liu G, Wu J, Yang H, Bao Q (2010) Codon usage patterns in Corynebacterium glutamicum: mutational bias, natural selection and amino acid conservation. Comp Funct Genomics 2010:343569

    PubMed Central  PubMed  Google Scholar 

  • Lv Y, Wu Z, Han S, Lin Y, Zheng S (2012) Construction of recombinant Corynebacterium glutamicum for l-threonine production. Biotechnol Bioprocess Eng 17(1):16–21

    Article  CAS  Google Scholar 

  • Malumbres M, Gil JA, Martin JF (1993) Codon preference in Corynebacteria. Gene 134(1):15–24

    Article  CAS  PubMed  Google Scholar 

  • Matsuda Y, Itaya H, Kikuchi Y, Beppu H, Jomantas JAV, Kutukova EA (2013) Methods for secretory production of protein. WO/2013/065839

  • Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, Marin L, Yu A, Lam C, Rodriguez C, Bennett G, Keasling JD, Endy D, Arkin AP (2013) Quantitative estimation of activity and quality for collections of functional genetic elements. Nat Methods 10(4):347–353

    Article  CAS  PubMed  Google Scholar 

  • Neuner A, Heinzle E (2011) Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering. Biotehcnol J 6(3):318–329

    CAS  Google Scholar 

  • Park JU, Jo JH, Kim YJ, Chung SS, Lee JH, Lee HH (2008) Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes. J Microbiol Biotechnol 18(4):639–647

    PubMed  Google Scholar 

  • Pátek M, Nešvera J (2013) Promoters and plasmid vectors of Corynebacterium glutamicum. In: Yukawa H, Inui M (eds) Corynebacterium glutamicum: biology and biotechnology. Springer, Heidelberg, pp 51–88

    Google Scholar 

  • Persson J, Lester P (2004) Purification of antibody and antibody fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent. Biotechnol Bioeng 87(3):424–434

    Article  CAS  PubMed  Google Scholar 

  • Pfleger BF, Pitera DJ, Smolke CD, Keasling JD (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24:1027–1032

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sundaram RK, Hurwitz I, Matthews S, Hoy E, Kurapati S, Crawford C, Sundaram P, Durvasula RV (2008) Expression of a functional single-chain antibody via Corynebacterium pseudodiphtheriticum. Eur J Clin Microbiol Infect Dis 27:617–622

    Article  CAS  PubMed  Google Scholar 

  • Teramoto H, Watanabe K, Suzuki N, Inui M, Yukawa H (2011) High yield secretion of heterologous proteins in Corynebacterium glutamicum using its own Tat-type signal sequence. Appl Microbiol Biotechnol 91:677–687

    Article  CAS  PubMed  Google Scholar 

  • Vertès AA (2013) Protein secretion systems of Corynebacterium glutamicum. In: Yukawa H, Inui M (eds) Corynebacterium glutamicum: biology and biotechnology. Springer, Heidelberg, pp 351–389

    Google Scholar 

  • Watanabe K, Teramoto H, Suzuki N, Inui M, Yukawa H (2013) Influence of SigB inactivation on Corynebacterium glutamicum protein secretion. Appl Microbiol Biotechnol 97(11):4917–4926

    Article  CAS  PubMed  Google Scholar 

  • Wendisch VF, Bott M, Kalinowski J, Oldiges M, Wiechert W (2006) Emerging Corynebacterium glutamicum systems biology. J Biotechnol 123(1):74–92

    Article  Google Scholar 

  • Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694(1):299–310

    Article  CAS  PubMed  Google Scholar 

  • Yim SS, An SJ, Kang M, Lee J, Jeong KJ (2013) Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol Bioeng 110(11):2959–2971

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (grant no. 2011–0031955) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (MSIP).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea

    Sung Sun Yim, Seul Ji An, Jae Woong Choi, Ae Jin Ryu & Ki Jun Jeong

  2. Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, Republic of Korea

    Ki Jun Jeong

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  1. Sung Sun Yim
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  2. Seul Ji An
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  3. Jae Woong Choi
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Correspondence to Ki Jun Jeong.

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Yim, S.S., An, S.J., Choi, J.W. et al. High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum . Appl Microbiol Biotechnol 98, 273–284 (2014). https://doi.org/10.1007/s00253-013-5315-x

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