Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process

Abstract

BackgroundThe production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein.ResultsIt was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation–refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilization process was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer.ConclusionsMaltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1053-9) contains supplementary material, which is available to authorized users.

The impact of technical failures during cultivation of an inclusion body process

“…We have shown that the maximum intracellular IB size varied between 500 and 700 nm [16], which resembles a ratio around 30% of IB per cell given the rough E. coli size estimation of 2 µm. Furthermore, the basic and cheap refolding with glycerol as single additive in deionized water resulted in a refolding yield of around 30% compared to ~ 49% [39] or 32.3% [35] for similar proteins in more complex buffers.…”

Production and immunogenicity of Fubc subunit protein redesigned from DENV envelope protein

“…The resulting pellet protein was washed 4 to 5 times with TE 50/20 (50 mM Tris pH 8.0 and 20 mM EDTA 20) buffer to remove impurities and extra salts. The remaining pellet was re-solubilised using mild denaturing agent such as 4.0 M urea and 5% n-propanol along with PBS buffer (pH 8.1) (Sarker et al 2019) and was centrifuged at 20,000 rpm for 10 min. The soluble protein fraction was initially purified by using Ni-NTA agarose beads and was confirmed by western blot using anti-His antibody.…”

“…Chaperone-assisted folding system did not help remarkably to express the Fubc recombinant protein in the soluble form. Therefore, the insoluble pellet fraction of Fubc protein was further utilised in mild solubilisation process (Sarker et al 2019) to recover soluble Fubc protein. Initially, the recovered soluble Fubc protein fraction was purified by using Ni-NTA agarose beads, and the purified protein band was confirmed by Western blot analysis using anti-His tag monoclonal antibody (Fig.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Batch mode refolding of solubilized IBs can be carried out using rapid or pulse dilution, by diafiltration and dialysis or by on-column chromatographic methods. In a recent study, it was also established that mild solubilization can be considered in terms of cost, time, and tag-free nature for the recovery of scFv from IBs (Sarker et al, 2019). The various methods used for refolding to recovery biologically active proteins have been reviewed previously (Rathore et al, 2013;Yamaguchi and Miyazaki, 2014).…”