The expression of recombinant proteins in E. coli includes various factors each of which plays a critical role sometimes. The typical procedure to produce heterologous proteins consists of the following steps: (1) construction of an overexpression plasmid by insertion of the target gene into a suitable vector, (2) transformation of a proper strain for protein production with the constructed plasmid, (3) growth of the host in a proper medium and the induction of the protein production at a proper timing, and (4) further growth to get the highest possible yield. We constructed an expression plasmid with an autoinducible promoter working in a general host that was not designed for protein production, and in a TB medium which is much easier to prepare compared to the sofisticated defined medium. This plasmid also contains a color indicator which turns red as the protein is produced. Since the plasmid works with general hosts, one can directly go to the production stage right after selecting the colony with the correctly constructed plasmid. With some experience, one can decide when to harvest the culture. We hope that this plasmid will help researchers to save their time and effort in a routine protein production procedure.
From left to right. Cell pellets. (a) Rosetta2(DE3)pLysS/pVP65KR-SacB(-) grown in (a) OE and (b) LB and induced with IPTG; (c) XL10-Gold/pVP65KR-gb-SacB(-) grown in TB overnight.