Bacterial Expression Systems
Introduction
Molecular biologists commonly design recombinant proteins which they can introduce through DNA plasmids containing inducible promoters upstream of the corresponding gene. Generally, E.coli BL21 cells are transformed with the appropriate plasmid, cultured shaking in selective LB medium at RT until the culture reaches OD (600nm) of ~ 0.6, at which time expression of recombinant proteins is induced by introducing IPTG into the cell culture.
T7 Promotor – IPTG method of induction
Expression Clones
E.coli BL21 cell strains are commonly used for recombinant protein expression. Several BL21 strains are available with distinct features that may be optimal depending on the proteins to be expressed. When expressing mammalian proteins in bacterial systems, one must consider that genomes of certain organisms may favour sequences with codons that occur infrequently in e.coli, thus exhausting the cell’s corresponding tRNAs upon induced expression. This may lead to frame shifts, codon skipping, misincorporations, truncated protein products, or complete lack of expression.
To circumvent the problem of codon bias, some BL21 strains are supplemented with extra copies of tRNA genes that are rare to E.coli. For example, extra copies of argU, ileY, and leuW tRNA genes, which recognize the AGA/AGG, AUA, and CUA codons respectively, support sequences from AT-rich genomes. Furthermore, protease deficiencies (ex. For Lon and OmpT protease) may be established to prevent product degradation for increased protein stability.
Considerations for Selection
Different strains of BL21 have different combinations of antibiotic resistance. In order to select for positive clones, a BL21 strain must be selected such that it does not contain the same antibiotic resistance gene as that present within the vector. The corresponding antibiotic must be supplemented in the media to select for positive clones with the vector. For example, if using a vector with chloramphenicol resistance, shuch as pACYC-Duet-1, e.coli BL21-D3-RIPL would be an inappropriate choice as the strain is already resistant for chloramphenicol, thus clones lacking the vector will survive.