Enhanced Solubility of Anti-HER2 scFv Using Bacterial Pelb Leader Sequence

Document Type : Research Paper


1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 bStudent Research Committee, Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Department of Biotechnology, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran;Pharmaceutical Science Research Center, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran.

4 aDepartment of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran;Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.



Single chain Fragment variable (scFv) is an antibody fragment consisting variable regions of heavy and light chains. scFvs enhance their penetrability into tissues while maintaining specific affinity and having low immunogenicity. Insoluble inclusion bodies are formed when scFvs are expressed in reducing bacterial cytoplasm. One strategy for obtaining functionally active scFv is to translocate the scFv into the oxidized environment of the periplasm where the possibility for disulfide bond formation is increased. This can be achieved by cloning the gene in a vector containing N-terminal pelB leader peptide that export foreign proteins to the periplasmic space. The aim of this study is to evaluate the influence of periplasmic localization using pelB leader peptide on the solubility of anti HER2-scFv.
Herein, anti HER2-scFv gene was cloned between NcoI and XhoI sites of pET22-b (+) containing pelB leader peptide and in same sites of pET28-b (+) (without pelB). The expression in BL21 (DE3) was induced using IPTG and was analyzed using SDS-PAGE and Western blot experiment. Then, the solubility of anti HER2-scFvin BL21 (DE3) containing both pET22- and pET28-(anti HER2-scFv) was determined.
The results of the present study demonstrated that anti HER2-scFv was expressed by both pET22-b (+) and pET28-b (+) vectors in BL21 (DE3). The proper expression of anti-HER2 scFv was confirmed by appearance of a  28 kDa band in Western blot analysis. The most anti HER2-scFv expression from BL21 containing pET28-(anti HER2-scFv) was achieved when it was induced by 0.25 mM IPTG at 37 C, 24 h post-induction. The ratio of soluble/insoluble anti HER2-scFv was significantly higher in BL21 containing pET22-(anti HER2-scFv) than in that containing pET28-(anti HER2-scFv). Totally, fusion of pelB signal sequence to anti HER2-scFv resulted in solubility enhancement. Therefore, production of functional anti HER2-scFv with proper disulfide bond can be achieved by directing the recombinant protein to periplasmic space using pelB signal peptide in pET22 (+) vector.


[1] Farajnia S, Ahmadzadeh V, Tanomand A, Veisi K, Khosroshahi SA, Rahbarnia L. Development trends for generation of single-chain antibody fragments. Immunopharmacol. Immunotoxicol. (2014) 36 (5): 297-308.
[2] Weisser NE, Hall JC. Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol. Adv. (2009) 27 (4): 502-520.
[3] Blažek D, Celer V. The production and application of single-chain antibody fragments. Folia. Microbiol. (Praha) (2003) 48 (5): 687-98.
[4] Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. ScFv antibody: principles and clinical application. Clin. Dev. Immunol. (2012)2012: 980250.
[5] Gopal GJ, Kumar A. Strategies for the production of recombinant protein in Escherichia coli. Protein J. (2013) 32 (6): 419-25.
[6] Zhu YQ, Tong WY, Wei DZ, Zhou F, Zhao JB. Environmental stimuli on the soluble expression of anti-human ovarian carcinoma× anti-human CD3 single-chain bispecific antibody in recombinant Escherichia coli. Biochem. Eng. J. (2007) 37 (2): 184-91.
[7] Terpe K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. (2006) 72 (2): 211.
[8] Kamionka M. Engineering of therapeutic proteins production in Escherichia coli. Curr. Pharm. Biotechnol. (2011) 12 (2): 268-74.
[9] Lindner R, Moosmann A, Dietrich A, Böttinger H, Kontermann R, Siemann-Herzberg M. Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli. J. Biotechnol. (2014) 192: 136-45.
[10] De Marco A. Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb. Cell Fact. (2009) 8 (1): 26.
[11] Mergulhao F, Summers DK, Monteiro GA. Mergulhao F, Summers DK, Monteiro GA. Biotechnol. Adv. (2005) 23 (3): 177-202.
[12] Hajighasemlou et al. Preparation of immunotoxin herceptin-botulinum and killing effects on two breast cancer cell lines. Asian Pac. J. Cancer Prev. (2015) 16: 5977-81.
[13] Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Science (1987) 235 (4785): 177-82.
[14] Akbari V, Mir Mohammad Sadeghi H, Jafrian-Dehkordi A, Abedi D, Chou CP. Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli. J. Ind. Microbiol. Biotechnol. (2014) 41 (6): 947-56.
[15] Cao et al. Single-chain antibody-based immunotoxins targeting Her2/neu: design optimization and impact of affinity on antitumor efficacy and off-target toxicity. Mol. Cancer Ther. (2012) 11(1): 143-53.
[16] Cao Y, Marks JD, Marks JW, Cheung LH, Kim S, Rosenblum MG. Construction and characterization of novel, recombinant immunotoxins targeting the Her2/neu oncogene product: in vitro and in vivo studies. Cancer Res. (2009) 69 (23): 8987-95.
[17] Park et al. Anti-HER2 immunoliposomes enhanced efficacy attributable to targeted delivery. Clin. Cancer Res. (2002) 8 (4): 1172-81.
[18] Park et al. Tumor targeting using anti-her2 immunoliposomes. J. Control Release (2001) 74 (1): 95-113.
[19] Novagen I. pET system manual. Novagen Madison, WI (2002).
[20] Heo MA, Kim SH, Kim SY, Kim YJ, Chung J, Oh MK, Lee SG. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli. Protein Expr. Purif. (2006) 47 (1): 203-9.
[21] Napathorn SC, Kuroki M. High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli. Anticancer Res. (2014) 34 (8): 3937-46.
[22] Naderi S, Alikhani MY, Karimi J, Shabab N, Mohamadi N, Jaliani HZ, Saidijam M. Cytoplasmic expression, optimization and catalytic activity evaluation of recombinant mature lysostaphin as an anti-staphylococcal therapeutic in Escherichia coli. Acta Med. Int. (2015) 2 (2): 72.
[23] Jaliani HZ, Farajnia S, Safdari Y, Mohammadi SA, Barzegar A, Talebi S. Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase. Adv. Pharm. Bull. (2014) 4 (3): 261.
[24] Drees JJ, Augustin LB, Mertensotto MJ, Schottel JL, Leonard AS, Saltzman DA. Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli. Protein Expr. Purif. (2014) 94: 60-6.
[25] Padiolleau-Lefèvre S, Débat H, Phichith D, Thomas D, Friboulet A, Avalle B. Expression of a functional scFv fragment of an anti-idiotypic antibody with a β-lactam hydrolytic activity. Immunol. Lett. (2006)103(1): 39-44.
[26] Tiwari A, Sankhyan A, Khanna N, Sinha S. Enhanced periplasmic expression of high affinity humanized scFv against Hepatitis B surface antigen by codon optimization. Protein Expr. Purif. (2010) 74 (2): 272-9.
[27] Cronin et al. Annual Report to the Nation on the Status of Cancer, part I. National cancer statistics. Cancer (2018) 124 (13): 2785-2800.
[28] Salamzadeh J, Kamalinejad M, Mofid B, Mortazavi SA, Sheikhlar A, Babaeian M. The Effect of Elaeagnus angustifolia L. Cream on Radiation-Induced Skin Reactions in Women with Breast Cancer; A Preliminary Clinical Trial. Iran. J. Pharm. Sci. (2017) 13 (2): 25-36.
[29] Harrison J, Keshavarz‐Moore E. Production of antibody fragments in Escherichia coli. Ann. N. Y. Acad. Sci. (1996) 782 (1): 143-58.
[30] Ahmadzadeh M, Farshdari F, Nematollahi L, Behdani M, Mohit E. Anti‑HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity. Mol. Biotechnol. (2020) 62: 18–30.