Expression and purification of soluble recombinant β-lactamases using Escherichia coli as expression host and pET-28a as cloning vector

Abstract

Background Due to its high expression capability, recombination of Escherichia coli and pET vector has become the bioengineering preferred expression system. Because β-lactamases mediate bacterial antimicrobial resistance, these enzymes have a substantial clinical impact. Using the E. coli expression system, several kinds of β-lactamases have been produced. However, previous studies have been focused on characterizing target β-lactamases, and the effects of cultivation and induction conditions on the expression efficiency of target enzymes were not addressed. Results Using pET-28a as the cloning vector and E. coli BL21(DE3) as the expression host, this study originally elucidated the effects of IPTG concentration, culture temperature, induction time, and restriction sites on recombinant β-lactamase expression. Moreover, the effects of the target protein length and the 6 × His-tag fusion position on enzyme purification were also explored, and consequently, this study yielded several important findings. (i) Only the signal peptide–detached recombinant β-lactamase could exist in a soluble form. (ii) Low-temperature induction was beneficial for soluble β-lactamase expression. (iii) The closer to the rbs the selected restriction site was, the more difficult it was to express soluble β-lactamase. (iv) The short-chain recombinant protein and the protein with His-tag fused at its C-terminus showed high affinity to the Ni2+ column. Conclusions Based on our findings, researchers can easily design an effective program for the high production of soluble recombinant β-lactamases to facilitate other related studies.

Design of a novel multi-epitopes vaccine against Escherichia fergusonii: a pan-proteome based in- silico approach

“…The optimized and improved sequence was cloned into a specific vector known as pet28+(a) (56). This vector is mostly used in cloning approach because it consists of T7 promoter, His-tag fusion (polyhistadine which facilitates easy purification of the expressed protein), multiple-cloning-site and a selectable marker (57). The resulting data from this step will provide precise data for experimentalists to enable industrial-level production.…”