J. Microbiol. Biotechnol. (2017), 27(5), 965–974 https://doi.org/10.4014/jmb.1702.02007 Research Article Review jmb Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis Wei Hu1†, Jun-Yan Xiang1†, Ping Kong1, Ling Liu1, Qiuhong Xie1,2,3*, and Hongyu Xiang1,2,3* 1School of Life Science, 2Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, 3National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun, Jilin 130012, P.R. China Received: February 6, 2017 Revised: February 28, 2017 The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein Accepted: March 9, 2017 receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be First published online the starting point for further engineering and application of the scFv. In this study, the March 9, 2017 abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and *Corresponding authors Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it Q.X. Phone: +86-431-85153832; was efficiently secreted by B. choshinensis. The optimized fermentation was determined using Fax: +86-431-85153832; the Plackett-Burman screening design and response surface methodology, under which the E-mail: [email protected] yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained H.X. Phone: +86-431-85153832; from the two expression systems were different. The antigen affinity, transition temperature, Fax: +86-431-85153832; and particle diameter size were 1.01E-07 M, 55.2 ± 0.3ºC, and 9.388 nm for the scFv expressed E-mail: [email protected] by B. choshinensis, and 4.53E-07 M, 52.5 ± 0.3ºC, and 13.54 nm for the scFv expressed by E. coli. †These authors contributed This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, equally to this work. which will boost its use in LOX-1-based therapy. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2017 by Keywords: Lectin-like oxidized low-density lipoprotein receptor-1, single-chain variable The Korean Society for Microbiology fragment antibody, Brevibacillus choshinensis, Escherichia coli, production methodology and Biotechnology Introduction (MAbs) [5]. Moreover, LOX-1 can be used for plaque imaging using MAbs and selective delivery of anti-atherosclerotic Lectin-like oxidized low-density lipoprotein receptor-1 agents [6]. (LOX-1), a multi-ligand scavenger receptor that was originally MAbs occupy a large portion of biopharmaceutical identified as the primary receptor for oxLDL, mediates a proteins [7]. There are mainly two disadvantages of using series of proatherogenic cellular responses implicated in the MAb molecules; one is the large molecular size, which the pathogenesis of atherosclerosis-related diseases [1, 2]. limits their penetration into diseased areas, and the other is These diseases continue to be a major cause of morbidity the binding of the crystallizable fragment (Fc) domain of and mortality in the world. LOX-1 basal expression is the MAbs to the cell surface Fc receptor, which limits their relatively low, and it is substantially up-regulated in circulation and mobility [8]. As a substitute for large intact multiple disease states [3]. LOX-1 expressed on the cell MAbs, scFvs have tremendous potential for use in surface can be proteolytically cleaved and released into the diagnosis and targeted therapy because of their specific circulation in a soluble form (sLOX-1). sLOX-1 has proved binding affinity to the antigen, their small size, superior to be a sensitive diagnostic biomarker of vascular pathology biodistribution, and blood clearance; additionally, they can [4]. In addition, recent evidence indicates that targeting be easily engineered or modified [9, 10]. Recently, an anti- LOX-1 is a reliable strategy for the therapy of vascular LOX-1 scFv has been developed for targeting LOX-1 [11], disease and that LOX-1-mediated proatherogenic effects which we believe has rich potential as a LOX-1 targeting can be inhibited by anti-LOX-1 monoclonal antibodies vector for immunodiagnostics, drug delivery, and potential May 2017 ⎪ Vol. 27⎪ No. 5 966 Hu et al. immunotherapy, that requires further research. However, extract, 0.5% beef extract, 0.001% FeSO4·7H2O, 0.001% MnSO4·4H2O, the minor yield of this scFv produced using the Free Style and 0.0001% ZnSO4·7H2O), and LB. 293 expression system limited its further engineering and application. Therefore, it is urgent to develop efficient Plasmid Construction methods for producing this scFv. The anti-LOX-1 scFv was designed on the basis of sequences of the variable heavy (V ) and light (V ) chains of the anti-human ScFv can be produced using a wide range of platforms, H L LOX-1 antibody [11], in the form of V −(Gly Ser) −V with a C- including production in the periplasm of prokaryotes [12]; H 4 3 L terminal peptide encoding the myc (myc) epitope and a in the endoplasmic reticulum of eukaryotes [13]; and in polyhistidine (6×His) metal-binding tag to facilitate the purification cell-free expression systems [14]. Of these expression and immunodetection of the gene product. The gene fragment systems, Escherichia coli is a widely used host that is was chemically synthesized and cloned into a pUC19 vector by suitable for expressing small non-glycosylated recombinant Beijing Genomic Institute (BGI, China), and the plasmid was antibody fragments, including scFv [15, 16]. In recent years, named pUC19-scFv. a promising host, Brevibacillus choshinensis, was developed Plasmid pUC19-scFv was used as the template for the PCR. To for antibody fragment production. Using B. choshinensis, we append the EcoRI and SalI restriction sites for cloning into the have successfully obtained secreted matrix metalloproteinase vectors pET-21a(+) and pET-23a(+), the primer sets 5’-CGG 26 [17], which expressed as inclusion bodies in E. coli hosts. AATTCGAAGTCAAACTGCTGGAATCTGG-3’ (forward) and 5’- In this study, the abundant expression of the anti-LOX-1 GCGTCGACTCATTAGTGGTGGTGATGATGGTGAGC-3’ (reverse) were used. For cloning into the vectors pET-20b(+) and pET- scFv was attempted using E. coli and B. choshinensis. The 27b(+), primer sets 5’-CGGAATTCCGAAGTCAAACTGCTGGAA optimization study was performed using Plackett-Burman TCTGG-3’ (forward) and the same reverse primer as mentioned screening design and the response surface methodology above were used. For cloning into the vector pNCMO2, primer (RSM). After that, the scFvs obtained from the two sets 5’-GCGTCGACGAAGTCAAACTGCTGGAATCTGG-3’ (forward) expression systems were purified using a two-step column and 5’-CGGAATTCTCATTAGTGGTGGTGATGATGGTGAGC-3’ chromatography purification progress. Finally, properties (reverse) were used. The resulting PCR products were digested of the purified anti-LOX-1 scFv proteins were studied. As with EcoRI and SalI, and then they were cloned into the vectors an ideal antibody for use in therapeutic purposes, it must digested using the corresponding restriction enzymes to form the have good pharmaceutical properties, such as antigen affinity expression plasmids. These expression plasmids were correspondingly and thermal stability. Results showed that B. choshinensis named pET21-scFv, pET23-scFv, pET20-scFv, pET27-scFv, and can act as an efficient host for the secreted expression of the pNC-scFv. anti-LOX-1 scFv in abundance. Expression of the Anti-LOX-1 scFv in E. coli Hosts E. coli strain BL21(DE3), Origami, Rosetta-gami 2(DE3), and Materials and Methods Rosetta-gami 2(DE3)pLysS were respectively transformed with the expression plasmids pET21-scFv, pET23-scFv, pET20-scFv, Bacterial Strains, Vectors, and Media and pET27-scFv, and then the transformants were cultured in LB The E. coli strains (Novagen, USA) BL21(DE3), Origami, Rosetta- medium containing the required antibiotic(s). The culture was gami 2(DE3), and Rosetta-gami 2(DE3)pLysS, and B. choshinensis shaken at 37ºC until the cell density (OD600) reached 0.4-0.6. Then, SP3 (Takara, Japan) were used for scFv expression. The vector the induction was conducted by adding 0.5 mM isopropyl-1-thio- pNCMO2, a B. choshinensis-E. coli shuttle vector obtained from β-D-galactopyranoside (IPTG). For optimization, cultivations were Takara, was used for the expression in B. choshinensis. The vectors performed using different IPTG concentrations (0.25, 0.5, 1, or (Novagen) pET-20b(+), pET-21a(+), pET-23a(+), and pET-27b(+) 2 mM) and temperatures (18ºC, 25ºC, 30ºC, or 37ºC). The cells were were used for expressing the scFv in E. coli. harvested by centrifugation, and the cell pellet was resuspended LB medium was used to culture E. coli. B. choshinensis strains in 50 mM sodium phosphate buffer (PBS, pH 7.4) containing 0.2 M used for the expression and secretion of anti-LOX-1 scFv was NaCl. For cell lysis, the cells were homogenized and disrupted by respectively grown in medium 5YC (3% glucose, 3% polypeptone, sonication. The lysates were centrifuged at 27,000 ×g for 30 min at 0.2% yeast extract, 0.01% MgCl ·6H O, 0.01% CaCl ·2H O, 0.001% 2 2 2 2 4ºC. FeSO4·7H2O, 0.001% MnSO4·4H2O, and 0.0001% ZnSO4·7H2O), B2 (0.5% glucose, 1% soytone, 2.5% yeast extract, 2.5% NaCl, and Secreted Expression of Anti-LOX-1 scFv in the B. choshinensis Host 0.1% K HPO ), 2SY (2% glucose, 4% soytone, 0.5% yeast extract, 2 4 B. choshinensis SP3 cells harboring the plasmid pNC-scFv were and 0.015% CaCl ·2H O), BTY (1% glucose, 1% polypeptone, 0.5% 2 2 cultured overnight at 37ºC in 2SY, BTY, LB, 5YC, B2, and/or TM yeast extract, 0.001% FeSO ·7H O, 0.001% MnSO ·4H O, and 0.