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Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner

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Abstract

Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.

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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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barcena J, Ochoa M, delaLuna S, Melero JA, Nieto A, Ortin J, Portela A (1994) Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro. J Virol 68:6900–6909

    CAS  PubMed  PubMed Central  Google Scholar 

  • Beaton AR, Krug RM (1986) Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. Proc Natl Acad Sci USA 83:6282–6286

    Article  CAS  PubMed  Google Scholar 

  • Biocca S, Cattaneo A (1995) Intracellular immunization: antibody targeting to subcellular compartments. Trends Cell Biol 5:248–252

    Article  CAS  PubMed  Google Scholar 

  • Boulo S, Akarsu H, Ruigrok RW, Baudin F (2007) Nuclear traffic of Influenza virus proteins and ribonucleo protein complexes. Virus Res. 124:12–21

    Article  CAS  PubMed  Google Scholar 

  • Chaisri U, Chaicumpa W (2018) Evolution of therapeutic antibodies, influenza virus biology, influenza, and influenza immunotherapy. Biomed Res Int 2018:9747549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cros JF, Palese P (2003) Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res. 95:3–12

    Article  CAS  PubMed  Google Scholar 

  • Devyani S, Asma S, Sandeep B, Atul KP, Rekha K, Richa CS, Anil P, Bramhadev P, Hare KP (2014) Development of single-chain Fv against the nucleoprotein of type A influenza virus and its use in ELISA. J Virol Methods 208:129–137

    Article  CAS  Google Scholar 

  • Eiryo K, Tokiko W, Ken F, Hideo G, Shinji W, Takeshi N, Yoshihiro K (2011) Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. J Virol Methods 173:1–6

    Article  CAS  Google Scholar 

  • Frenzel A, Hust M, Schirrmann T (2013) Expression of recombinant antibodies. Front Immunol 4:217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J (2005) The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci USA 102:18590–18595

    Article  CAS  PubMed  Google Scholar 

  • Gao Y, Sun L, Dong J, Xu X, ShuY CM, Yin L, Liang Z, Jin Q (2006) Rapid identification of small interfering RNA that can effectively inhibit the replication of multiple influenza B virus strains. Antivir Ther 11:431–438

    CAS  PubMed  Google Scholar 

  • Ge Q, McManus MT, Nguyen T, Shen CH, Sharp PA, Eisen HN, Chen J (2003) RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA 100:2718–2723

    Article  CAS  PubMed  Google Scholar 

  • Hao SM, Li Q, Wang CY, Wang HL, Yang ST, Xia XZ (2009) Cloning and expression of single chain Fv antibodies against H5N1 avian influenza virus hemagglutinin. Bing Du Xue Bao 25:63–67

    CAS  PubMed  Google Scholar 

  • Kudou M, Ejima D, Sato H, Yumioka R, Arakawa T, Tsumoto K (2011) Refolding single-chain antibody (scFv) using lauroyl-L-glutamate as a solubilization detergent and arginine as a refolding additive. Protein Expr Purif 77:68–74

    Article  CAS  PubMed  Google Scholar 

  • Li TW, Cheng SF, Tseng YT, Yang YC, Liu WC, Wang SC, Chou MJ, Lin YJ, Wang Y, Hsiao PW, Wu SC, Chang DK (2016) Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2). Arch Virol 161:19–31

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Qin K, Meng G, Zhang J, Zhou J, Zhao G, Luo M, Zheng X (2013) Structural and functional characterization of K339T substitution identified in the PB2 subunit cap-binding pocket of influenza A virus. J Biol Chem 288:11013–11023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Maneewatch S, Thanongsaksrikul J, Songserm T, Thueng-In K, Kulkeaw K, Thathaisong U, Srimanote P, Tongtawe P, Tapchaisri P, Chaicumpa W (2009) Human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza A virus subtype H5N1. Antivir Ther 14:221–230

    CAS  PubMed  Google Scholar 

  • Nicholls PJ, Johnson VG, Blanford MD, Andrew SM (1993) An improved method for generating single-chain antibodies from hybridomas. J Immunol Methods 165:81–91

    Article  CAS  PubMed  Google Scholar 

  • Palese P (1977) The genes of influenza virus. Cell 10:1–10

    Article  CAS  PubMed  Google Scholar 

  • Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88

    Article  CAS  PubMed  Google Scholar 

  • Popplewell AG, Sehdev M, Spitali M, Weir AN (2005) Expression of antibody fragments by periplasmic secretion in Escherichia coli. Methods Mol Biol 308:17–30

    CAS  PubMed  Google Scholar 

  • Poungpair O, Chaicumpa W, Kulkeaw K, Maneewatch S, Theung K, Srimanote P, Tongtawe P, Songserm T, Lekcharoensuk P, Tapchaisri P (2009) Human single chain monoclonal antibody that recognizes matrix protein of heterologous influenza A virus subtypes. J Virol Methods 159:105–111

    Article  CAS  PubMed  Google Scholar 

  • Sapats SI, Heine HG, Trinidad L, Gould GJ, Foord AJ, Doolan SG, Prowse S, Ignjatovic J (2003) Generation of chicken single chain antibody variable fragments (scFv) that differentiate and neutralize infectious bursal disease virus (IBDV). Arch Virol 148:497–515

    Article  CAS  PubMed  Google Scholar 

  • Schaefer JV, Plückthun A (2010) Improving expression of scFv fragments by co-expression of periplasmic chaperones. In: Kontermann R, Dübel S (eds) Antibody engineering. Springer, Berlin, Heidelberg

  • Shimoura O (2006) Discovery of green fluorescent protein (GFP) (Nobel Lecture). Angew Chem Int Ed Engl 48:5590–5602

    Article  CAS  Google Scholar 

  • Short KR, Kroeze EJ, Fouchier RA, Kuiken T (2014) Pathogenesis of influenza- induced acute respiratory distress syndrome. Lancet Infect Dis 14:57–69

    Article  CAS  PubMed  Google Scholar 

  • Singh A, Upadhyay V, Upadyay AK, Singh SM, Panda AK (2015) Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial Cell Factories 14:41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Su Y, Zou Z, Feng S, Zhou P, Cao L (2007) The acidity of protein fusion partners predominantly determines the efficacy to improve the solubility of the target proteins expressed in Escherichia coli. J Biotechnol 129:373–382

    Article  CAS  PubMed  Google Scholar 

  • Subbarao EK, London W, Murphy BR (1993) A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol 67:1761–1764

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sun W, Xie J, Lin H, Mi S, Li Z, Hua F, Hu Z (2012) A combined strategy improves the solubility of aggregation-prone single-chain variable fragment antibodies. Protein Expr Purif 83:21–29

    Article  CAS  PubMed  Google Scholar 

  • Talavera D, Castillo AM, Dominguez MC, Gutierrez AE, Meza I (2004) IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers. J Gen Virol 85:1801–1813

  • Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    Article  CAS  PubMed  Google Scholar 

  • Umetsu M, Tsumoto K, Hara M, Ashish K, Goda S, Adschiri T, Kumagai I (2003) How additives influence the refolding of immunoglobulinfolded proteins in a stepwise dialysis system. Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment. J Biol Chem 278:8979–8987

    Article  CAS  PubMed  Google Scholar 

  • Webby RJ, Webster RG (2001) Emergence of influenza A viruses. Philos Trans R Soc Lond B Biol Sci 356:1817–1828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yodsheewan R, Maneewatch S, Srimanote P, Thueng-in K, Songserm T, Dong-din-on F, Bangphoomi K, Sookrung N, Choowongkomon K, Chaicumpa W (2013) Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes. Antiviral Res 100:226–237

  • Yokota T, Milenic DE, Whitlow M, Schlom J (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 52:3402–3408

    CAS  PubMed  Google Scholar 

  • Yusakul G, Sakamoto S, Nuntawong P, Tanaka H, Morimoto S (2018) Different expression systems resulted in varied binding properties of anti-paclitaxel single-chain variable fragment antibody clone 1C2. J Nat Med 72:310–316

    Article  CAS  PubMed  Google Scholar 

  • Zhang Z, Tang R, Zhu D, Wang W, Yi L, Ma L (2017) Non-peptide guided auto-secretion of recombinant proteins by superfolder green fluorescent protein in Escherichia coli. Sci Rep 7:6990

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang F, Chen Y, Ke Y, Zhang L, Zhang B, Yang L, Zhu J (2019) Single chain fragment variable (scFv) antibodies targeting the spike protein of porcine epidemic diarrhea virus provide protection against viral infection in piglets. Viruses 11:58

    Article  CAS  PubMed Central  Google Scholar 

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Funding

The work was supported by National Natural Science Foundation of China (Grant No. 31672561).

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Author notes

  1. Min Liu, Bin Wang and Fei Wang contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China

    Min Liu, Bin Wang, Fei Wang, Zhi Yang, Dan Gao, Chenyao Zhang, Lixin Ma & Xiaolan Yu

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  1. Min Liu
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  2. Bin Wang
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  3. Fei Wang
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  4. Zhi Yang
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  5. Dan Gao
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  6. Chenyao Zhang
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  7. Lixin Ma
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  8. Xiaolan Yu
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Contributions

Min Liu, Bin Wang, and Fei Wang performed the experiments and analyzed the data. Min Liu wrote the manuscript. Zhi Yang, Dan Gao, and Chenyao Zhang carried out the experiments. Xiaolan Yu and Lixin Ma designed the study and revised the manuscript.

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Correspondence to Lixin Ma or Xiaolan Yu.

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Liu, M., Wang, B., Wang, F. et al. Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner. Appl Microbiol Biotechnol 103, 6071–6079 (2019). https://doi.org/10.1007/s00253-019-09925-6

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  • DOI: https://doi.org/10.1007/s00253-019-09925-6

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