Escherichia coli ( E. coli ) is often used as a host to produce a variety of recombinant proteins. However, the direct production of soluble and active protein containing disulfide bonds in E. coli remains a major challenge. In this study, we established a new, innovative soluble protein expression system based on the synergistic action of elastin-like polypeptide (ELP) and intein. Kinds of proteins including nanobodies, hIL-3 (Human Interleukin-3) analogue and AVN (the N terminal of A. ventricosus MiSp) with amino acid sequences that are normally express as insoluble form or difficult to express could be successfully expressed in soluble form in E. coli with this system. The soluble expression of the target proteins in the cytoplasm was achieved by the fusion of the solubility enhancer ELP, and the fusion of intein allowed the target proteins to be directly separated from the solubility enhancers in a mild enzyme-free environment. Through a one or two-step operation, nanobodies with high affinity activity, IL-3 analogue with bioactivity and soluble AVN were easily purified. This simple and efficient soluble expression system could thus be widely applied to facilitate the expression of difficult-to-express proteins.
Yiwen Zhang,Yang Guo,Liang Song,Wenshuai Liu,Rui Nian,Xiying Fan
This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrap TM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli . This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli . Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.
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