Extracellular production of functional single-chain variable fragment against aflatoxin B₁usingEscherichia coli

Abstract

scFv‐BM3 is a single‐chain variable fragment (scFv) against aflatoxin B1 (AFB1) engineered by affinity maturation and site‐directed mutagenesis, and thus has a 31‐fold higher affinity than its wild‐type. To apply scFv‐BM3 to immunological detection of AFB1, periplasmic expression in Escherichia coli was attempted to produce a functional form of scFv‐BM3. scFv‐BM3 accumulated as inactive aggregates in the cells. However, it was found that scFv‐BM3 secreted into the culture medium had binding activity to AFB1. Expression conditions for scFv‐BM3 were further manipulated to enhance secretion into the culture medium. This extracellular secretion of functional scFv‐BM3 was significantly improved by supplementation with Triton X‐100 and optimization of expression conditions. The scFv‐BM3 purified from the culture medium exhibited a typical antiparallel β‐sheet structure and adopted a proper conformation to bind AFB1 with high affinity and specificity in various biophysical and biochemical analyses. Significance and Impact of the Study Single‐chain variable fragments (scFvs) are recombinant antibodies that are difficult to produce as a functional form in Escherichia coli. This study demonstrates the production of functional scFvs against aflatoxin B1 (AFB1) (scFv‐BM3) using Escherichia coli by extracellular secretion. While periplasmic expression of scFv‐BM3 resulted in formation of inactive aggregates in E. coli, the scFv‐BM3 secreted into the culture medium adopted a properly folded structure for specific binding to AFB1. This study promotes the application of functional scFv‐BM3 to the immunological detection of AFB1 in biotechnology fields.

Recent Progress in Rapid Determination of Mycotoxins Based on Emerging Biorecognition Molecules: A Review

“…RAbs have been developed and applied in ELISA for AFB1 [268,269], OTA [270,271], ZEN [95,110], DON [171,272], FB1 [273,274], CIT [275], and T-2/HT-2 toxins [94]. One of the major advantages of the rAb-based ELISA is that rAbs-reporter fusions can be expressed directly based on genetic engineering, which eliminates the chemical synthesis of antibody-reporter or use of commercial secondary antibody.…”

Single‐chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety

“…Biotoxins are toxic chemical substances produced by natural organisms that not only endanger human health but also cause serious losses in agriculture, animal husbandry, and aquaculture. In recent decades, a vast plethora of scFv antibodies for monitoring applications has been generated via phage display against biotoxins, including microcystins (Xu, Liu, et al., 2019; Xu, Miao, et al., 2019; Zhang, He, Zhao, Feng, et al., 2016), aflatoxin B1 (Min et al., 2011; Min et al., 2015; Na et al., 2019; Rangnoi et al., 2011, 2018), Bacillus thuringiensis cry toxins (Dong, Gao, Bo, et al., 2020; Dong, Gao, Guan, et al., 2020; Y. Wang et al., 2012; Z. L. Xu et al., 2017; Zhang et al., 2014; Zhong et al., 2018), and zearalenone (Edupuganti et al., 2013; Sompunga et al., 2019) (Table 4).…”

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

“…Beyond pelB, other N-terminal signal sequences derived from the outer membrane protein A (OmpA) or from alkaline phosphatase A (PhoA) have been utilized to transport antibody-like fragments to the periplasmic space of E. coli via the Sec pathway and the SRP-dependent pathway [146,147,156]. The 22 amino acids long PelB (pectate lyase B) signal sequence from Erwinia carotovora [157] is the most frequently used for transportation of the Fabs [88,96] and scFvs [30,101,158,159]. Recently, the expression of an scFv antibody fragment has been used as a showcase for validating the efficacy of the novel vector pSAR-2 containing the pelB leader sequence and the rhamnose-inducible expression promoter, obtaining a yield of 1.2 g/L [71].…”

“…Indeed, while glycine induces the swelling of E. coli cells and enlargement of the periplasmic space by interfering with the synthesis of peptidoglycans, Triton X-100 disrupts the integrity of the outer membrane [174]. In this way, a functional recombinant scFv has been efficiently recovered (2.86 mg/L) from the extracellular medium by adding 0.25% Triton X-100 [159]. An experimental setting for the efficient secretion of Fab fragments in E. coli in shake flasks has been recently described by Luo and coworkers [160].…”