pET-28a(+)

Due to its high expression ability, recombination between E. coli and pET vector has become the preferred expression system for bioengineering. Because β-lactamases mediate bacterial antimicrobial resistance, these enzymes have significant clinical impact. Several β-lactamases have been produced using E. coli expression systems. This study used pET-28a as the cloning vector and E. coli BL21 (DE3) as the expression host. It was the first time to elucidate the effects of IPTG concentration, culture temperature, induction time and enzyme cleavage site on the expression of recombinant β-lactamase. This study made some important findings. (i) Only recombinant β-lactamase with isolated signal peptide can exist in soluble form. (ii) Low temperature induction is beneficial to the expression of soluble β-lactamase. (iii) The closer the selected restriction site is to the RBS, the more difficult it will be to express the soluble β-lactamase. (iv) Short-chain recombinant proteins and proteins with C-terminal His tags fused show high affinity to the Ni²⁺ column.

In this study, two extended-spectrum β-lactamases, SHV-1 and TEM-1, encoded by the blaSHV-1 and blaTEM-1 genes, respectively, were selected as expression targets. Three recombinant pET-28a plasmids were generated for each expression target. (1) Recombinant plasmid 1: synthesize the complete blaSHV-1 or blaTEM-1 and insert it between the BamHI and EcoRI restriction sites to generate recombinant plasmids pET-28a–blaSHV-1/6230 bp and pET-28a–blaTEM-1/6230 bp (Figure 1A). (2) Recombinant plasmid 2: insert TAA-removed blaSHV-1 or blaTEM-1 between the NcoI and XhoI restriction sites to generate pET-28a–blaSHV-1/6097 bp and pET-28a–blaTEM-1/6097 bp plasmids (Figure 1B). (3) Recombinant plasmid 3: insert the deleted signal peptide coding region blaSHV-1 or blaTEM-1 between the NdeI and XhoI sites to generate pET-28a–blaSHV-1/6094 bp and pET-28a–blaTEM-1/6088 bp plasmids (Figure 1C).

Figure 1. Expression and purification of soluble recombinant β-lactamase using Escherichia coli as the expression host and pET-28a as the cloning vector. (Li, Lele, et al. 2022)