Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

Abstract

AbstractSingle-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.