Abstract
Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.
Highlights
In the past two decades, different technologies and expression platforms have been used for the successful production of recombinant antibody fragments, including single-chain variable fragment, fragment antigen binding (Fab), and nanobody, as new classes of therapeutic monoclonal antibodies (Gupta and Shukla 2017)
The periplasmic yield of antibody fragments is limited due to small volume of the E.coli periplasmic space but production of the scFv molecules in the periplasm offers a great number of advantages including: facilitating disulfide bond formation and proper folding, avoiding the intracellular protein degradation by proteases, and selective releasing the periplasmic scFvs with low host proteins and DNA contaminations resulting in less purification challenges during the product recovery (Gupta and Shukla 2016, 2017; Kasli et al 2019)
In the present study, we aimed to develop an optimum and generic approach for the lab-scale TES extraction of scFv antibody fragments from the periplasmic space of E. coli HB2151, which is being often used for the expression of soluble scFvs isolated from a phage antibody library
Summary
In the past two decades, different technologies and expression platforms have been used for the successful production of recombinant antibody fragments, including single-chain variable fragment (scFv), fragment antigen binding (Fab), and nanobody, as new classes of therapeutic monoclonal antibodies (Gupta and Shukla 2017). An increasing variety of expression systems have been developed ranging from bacteria, insects, yeasts, mammalian cells to transgenic plants and animals Of these expression systems, E. coli is the most popular used system for the production of scFv antibody fragments which take advantage of high productivity, straightforward cloning procedures, and the lowest manufacturing costs (Ahmad et al 2012; Gupta et al 2017). The periplasmic yield of antibody fragments is limited due to small volume of the E.coli periplasmic space but production of the scFv molecules in the periplasm offers a great number of advantages including: facilitating disulfide bond formation and proper folding, avoiding the intracellular protein degradation by proteases, and selective releasing the periplasmic scFvs with low host proteins and DNA contaminations resulting in less purification challenges during the product recovery (Gupta and Shukla 2016, 2017; Kasli et al 2019)
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