E coli Expression Host Selection
There are many expression hosts available for E. coli. The efficiency of protein expression results from appropriate selection of expression host. All of these hosts have the advantages and disadvantages. Invariably, the initial expression of the protein expression is analyzed by BL21(DE3) or derivatives of K-12 lineage strains. A couple of major characteristics of BL21 cells are; they are deprived of Lon protease which degrades many foreign proteins [33]. K-12 lineages are also used for checking the basic protein expression. The AD494 and Origami strains are trxB mutants which are capable enhancing the disulfide bond formation in the cytoplasm [34]. Another starin of K-12 lineage is HMS174, a recA mutant version which has a positive effect on plasmid stability [35].
Large number of bacterial hosts have been selected and tested for efficient expression of proteins. Some of the strains are modified to improve recombinant protein expression. These strains are generally defective in protease such as Lon or other outer membrane protease, OmpT. The preferred choice of expression hosts are BL21 and its derivatives. Some of the hosts’ strains are discussed in detail [36,37].
BL21(DE3): This strain is one of the widely used strain to check the basic protein expression in E. coli. Chromosomal DE3 prophase expresses T7 RNA polymerase under control of lacUV5 promoter. It is noticed that BL21 derivatives lack Lon and OmpT proteases which will stabilize expression of some recombinant proteins. While using this strain it is estimated that there is a chance of significant basal T7 expression or leaky expression. Addition of 1% glucose to the growth medium will reduce the leaky expression. Even though, addition of 1% glucose is recommended for toxic clones it can be applied to several other clones too which have leaky expressions.
BL21(DE3) pLysS: This strain has similar characteristics of BL21(DE3). However, pLysS produces wild-type T7 lysozyme to reduce basal T7 expression of the gene of interest.
BL21(DE3)pLysS strain is compatible with plasmids containing the ColE1 or pMB1 origin. Culturing of BL21(DE3)pLysS requires Chloramphenicol.
Lemo21(DE3): pLemo plasmid produces amidase negative T7 lysozyme (lysY) from a tunable promoter (Prha). This strain is compatible with plasmids containing the ColE1 or pMB1 origin. Most of the pET vectors are compatible with this strain. Chloramphenicol is required to maintain this strain.
BL21-AI: T7 RNA polymerase gene is controlled by the ParaBAD promoter. While using this strain, if pET vectors are used IPTG is also required for induction to titrate LacI repressor away from the T7- lac promoter on the vector. This strain is recommended while using pDEST vectors
TOP10: These are K - 12 strains that do not metabolize L-arabinose but may provide slight improvement when expressing membrane protein directly from ParaBAD. The strain is suitable for pBAD vectors
C41(DE3): These strains are derivatives of BL21(DE3) which has lower levels of T7 RNA polymerase under non-inducing and inducing conditions. In this strain, glucose addition is not necessary as with BL21(DE3).
Tuner(DE3): This strain is a lacZY derivative of BL21. The lac permease mutation (lacY1) allows uniform entry of IPTG into all cells in the population, which produces a concentration - dependent level of induction. The modified version of this strain is Tuner (DE3) pLysS. This is to expresses T7 lysozyme to control T7 expression in addition to the lac permease mutation.
Rosetta2 and Rosetta pLysS: Rosetta host strains are BL21 lacZY(Tuner) derivatives designed to enhance the expression of proteins which contains rare codons used in E.coli. These strains express tRNAs for rare codons on a compatible CamR plasmid. In case of pLysS strain, the rare tRNA genes and T7 lysozyme gene are carried by the same plasmid.
BL21 CodonPlus RIL and CodonPlus(DE3)–RIL/RIPL: BL21- Codon Plus strains are engineered to contain extra copies of genes that encode tRNAs which frequently limit the translation of heterologous proteins in E. coli. BL21-CodonPlus-RIL and BL21-CodonPlus (DE3)- RIL cells contain extra copies of the argU, ileY, and leuW tRNA genes. These genes encode tRNAs that recognize the arginine codons AGA and AGG, the isoleucine codon AUA, and the leucine codon CUA, respectively. The CodonPlusRIL strains have available the tRNAs that most frequently restrict translation of heterologous proteins from organisms that have AT-rich genomes. BL21-CodonPlus-RP and BL21-CodonPlus(DE3)-RP cells contain extra copies of the argU and proL genes. These genes encode tRNAs that recognize the arginine codons AGA and AGG and the proline codon CCC, respectively. The CodonPlus-RP strains have available the tRNAs that most frequently restrict translation of heterologous proteins of organisms that have GCrich genomes. The BL21-CodonPlus (DE3)-RIPL cells contain extra copies of the argU, ileY, and leuW as well as the proL tRNA genes. This strain rescues expression of heterologous proteins from organisms that have either AT- or GC-rich genomes.