Background

Due to its clear genetic background, easy manipulation, and cost-effective production, E. coli is widely employed for exogenous gene expression, especially those of lower molecular weight and simpler conformational structures (Gupta and Shukla, 2017). The expression of foreign proteins in E. coli is mainly divided into three categories, inclusion bodies expression, intracellular soluble expression in periplasmic space, and extracellular secretion into medium (Jalalirad, 2013; Gupta and Shukla, 2017; Zhou et al., 2018). At present, most of mammalian-sourced foreign proteins are expressed in E. coli intracellular region, either in the form of inclusion bodies or soluble. But both forms have their respective disadvantages for subsequent processes. The inclusion bodies need to be tediously denatured and renatured to recover the target proteins with correct refolded structures, and the processes tend to cause reduction in the biological activity and yield (Panda et al., 2003; Nelson and Reichert, 2009). The periplasmic space of E. coli can provide an oxidative environment conducive to the formation of disulfide bonds, but the yield is usually limited by the capacity of the periplasmic space as well as the leading capability of signal peptides (Lobstein et al., 2012; Ellis et al., 2017). Compared with the two processes above, extracellular protein expression is not restricted to the intracellular space, allows convenient enrichment of target proteins with right structures, thus simplifies the downstream purification processes (Zhou et al., 2018).

Fab fragment is of smaller size than full-length antibody, and does not require post-translational modification such as glycosylation modifications, it is particularly suitable to be produced in E. coli (Walsh and Jefferis, 2006; Rezaie et al., 2017). We recently reported a method for efficient extracellular expression and purification of Fabs from E. coli, which has been optimized for many parameters (Luo et al., 2019a). Here, we provide a detailed protocol using anti-VEGF Fab as a model protein, since the anti-VEGF Fab Ranibizumab was the first approved Fab drug on the market (Danyliv et al., 2017). The process consists of three main parts: A) construction of pPhoA-Fab expression vector and transfection of host strain BL21(DE3); B) secretory soluble expression of Fab in E. coli; and C) affinity chromatography purification of Fab. We investigated the combinations with different promoters (phoA and T7) and leader peptides (STII and pelB), among them phoA-STII showed the highest yield in mass and secretion efficiency. The vector was a previously engineered pRSF (Augustine et al., 2016) plasmid containing phoA promoter, hereinafter referred to as the pPhoA plasmid. The secretory expression was induced by phosphate starvation to stimulate the function of phoA promoter (Wang et al., 2005). For affinity purification of Fab, the resin should be selected according to species and light chain types (Kappa or Lambda chain). For anti-VEGF Fab, which has human Kappa light chain, we used a prepacked Capto L column to purify it (Ulmer et al., 2019). After purification, the final product could be further analyzed for purity, yield, and bioactivities.

Generally, secretory expression in E. coli is the most suitable process for producing disulfide bond-containing antibody fragments such as Fab, Fab’, and (Fab’)₂ (Ellis et al., 2017). In comparison with previous studies about extracellular expression of Fabs, our work is superior for a considerably reduced time and cost and simplified purification process, due to the use of different expression cassette designs (two separate expression cassettes, phoA promoter, STII leader sequence), host strains (BL21[DE3]) and fermentation conditions (phosphate starvation, low temperature). We have applied the protocol to prepare five Fab fragments which have been marketed successfully (anti IGF1R, anti-Her2, anti-VEGF, anti-RANKL and anti-PD-1) with different types of IgG1/IgG2 or human/humanized structures to cover as wide as possible range of Fab fragments. The results demonstrated that they were all expressed in soluble expression, and the fractions in culture medium were more than the intracellular soluble fractions or inclusion bodies content. By one-step affinity chromatography, the purity of the Fabs reached above 94%, and all products were of correct molecular weight as well as full bioactivity against their antigens. To the best of our knowledge, the current protocol is a universal technique for efficient extracellular expression, secretion and purification of Fabs in E. coli.