New tools for recombinant protein production in Escherichia coli: A 5-year update (1)
One derivative of B-lines, BL21(DE3) has become the preferred host for recombinant protein production. Genome sequencing of strains of the B line has helped understand the molecular basis of useful phenotypes for heterologous protein synthesis. common E. coli strains have a doubling time of about 20 minutes. Cultivation at pH 7.5–8.5 improves the production of recombinant proteins by lowering acetate stress. Deficiency in key proteolytic systems in E. coli B can extend the lifetime of recombinant proteins in certain cases. An IS186 insertion in the promoter of the lon gene eliminates this major protease. The BL21(DE3) strain carries a copy of phage T7 RNA poly- merase (T7RNAP) under control of the lacUV5 promoter. Genes of interest are cloned under control of a T7 promoter in expression plasmids, and protein production begins upon addition of the gratuitous inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). This system pro- vides the user with full control of the induction of protein synthesis with high selectivity and activity.
For BL21(DE3) and derivatives, strain engineering has advanced the capabilities of E. coli as a cell factory. The BL21(DE3) pLysS strain contains a plasmid (pLysS) with the gene for the T7 lysozyme, a natural inhibitor of T7RNAP. The Duet series of plasmids from Novagen allows for cloning two coding sequences under separate T7 promoters in the same plasmid. Up to eight proteins can be coexpressed using compatible Duet plasmids. The Marionette strains.24 In these cell lines (available in MG1655, DH10B, and BL21 backgrounds), the genes for 12 different genetic regulators were inserted in the genome.
The expression of toxic recombinant proteins causes cell death or poor growth. So, growth of IPTG-resistant colonies may indicate that genome modification in that strain allowed for gene expression of the offending protein. The popular C41(DE3) and C43(DE3) strains were isolated in this way. The isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3), which showed better yields for the production of membrane proteins than C41(DE3) and C43(DE3).
When using the BL21(DE3)/pET vectors system, the levels of recombinant proteins are difficult to manipulate, as IPTG is a potent inducer even at very low concentrations.
Plasmid maintenance is ensured by including a selection marker. Commonly, plasmids contain genes conferring antibiotic resistance. Media supplementation with antibiotics is simple, cost-effective, and convenient and is by far the most common strategy for protein synthesis at a lab scale.
The construction of high-copy number expression plasmids with increased stability has been described. Using a par locus, a cis-acting locus that allows stable plasmid inheritance, to assure retention of the plasmid pAR- KanI. After 8 hours of induction, almost all cells maintained the vector in the absence of antibiotic. In con- trast, if cells bearing the commercial pET28 vector are grown in the absence of antibiotic, only 5% retain the plasmid.
Fusion of the protein of interest to affinity tags and fusion partners markedly facilitate downstream processing. Commercial antibodies are available for many of the tags (for example, anti-His tag, anti-FLAG, anti-StrepII tag, to name a few) allowing for easy detection of the tagged protein by western blot. Typical fusion partners include glutathione S-transferase (GST), maltose binding protein (MBP), and members of the small ubiquitin-like modifier (SUMO) family of proteins. The addition of the sequence coding for serine-lysine-isoleucine-lysine (SKIK tag) after the initial N-terminal methionine codon mark- edly improves the expression of recombinant proteins. The NT-11 tag, derived from the first 11 amino acid residues of a duplicated carbonic anhydrase from Dunaliella.53 The NT-11 tag enhanced the soluble production of all tested proteins, which were also His-tagged. A 34-amino acid heparin-binding affinity tag (HB-tag) tagged proteins can be purified using a heparin-agarose resin under naturing or denaturing conditions. Tagged proteins can be eluted by a simple NaCl gradient.
Inclusion bodies (IBs) formation allows high product yield and purity and the production of toxic proteins in denaturing conditions.
Autoinduction media consists of at least two carbon sources: glucose and lactose (glycerol is also added to increase yields). Glucose is the preferred carbon source, and once it is depleted (typically during exponential growth), lactose starts being consumed. Using autoinduction media results in which higher bacterial densities are achieved, the time point of induction is highly reproducible, and stopping the culture for IPTG addition is no longer needed.
