PEPCF expresses proteins in bacteria, insect and mammalian cells and uses a variety of chromatographic and biophysical techniques for protein purification and characterization.
Many E. coli expression strains are derivatives from E. coli BL21(DE3). This strain is deficient in the lon and ompT proteases, which reduces the proteolytic activity. It also has a lysogenic λ prophage, which contains the T7 RNA polymerase gene under control of the lacUV5 promotor. This allows IPTG-inducible expression of genes under control of the T7 promotor (e.g. in pET vectors). Various derivative strains exist, which all have their own characteristics that might make them more suitable for the expression of specific subsets of proteins. Below you can find an overview of useful features of different E. coli expression strains.
The genetic code is degenerate, which means that one amino acid can be encoded by different codons. In different species some of the codons that encode the same amino acid are used more frequently than others, which is reflected in the genomic tRNA pool. If your gene of interest comes from a species with a very different codon usage than E. coli, this can lead to very low expression levels. One solution is to codon optimize your gene of interest (e.g. by ordering a codon-optimized synthetic gene). Another solution would be to use an E. coli strain with extra copies of rare tRNA’s for the expression of your protein.
BL21(DE3) codon + RIL: extra tRNA’s for rare codons for Arg (AGG/AGA), Ile (AUA) and Leu (CUA)
BL21(DE3) codon + RP: extra tRNA’s for rare codons for Arg (AGG/AGA) and Pro (CCC)
BL21(DE3) codon + RIPL: extra tRNA’s for rare codons for Arg (AGG/AGA), Ile (AUA), Pro (CCC) and Leu (CUA)
Rosetta(DE3): extra tRNA’s for rare codons for Arg (AGG/AGA), Ile (AUA), Leu (CUA), Pro (CCC) and Gly (GGA) → plasmid pRARE
Rosetta2(DE3): extra tRNA’s for rare codons for Arg (AGG/AGA/CGG), Ile (AUA), Leu (CUA), Pro (CCC) and Gly (GGA) → plasmid pRARE2
C41(DE3) and C43(DE3): these strains have some genetic mutations in the lacUV5 promoter region upstream of the T7 RNA polymerase gene, which allows them to produce levels of functional protein that would otherwise be toxic to the cell. They are often used for the expression of membrane proteins in E. coli as well.
BL21(DE3) pLysS/pLysE: the pLysS plasmid encodes the T7 lysozyme, which inhibits the T7 RNA polymerase
BL21-AI: the T7 RNA polymerase is under control of the araBAD promoter, which can be regulated very tightly. The promoter is repressed in the absence of arabinose and adding glucose further represses expression from the araBAD promoter by reducing the levels of cAMP.
AD94: mutations in trxB
Origami(DE3): mutations in trxB and gor → less reducing cytoplasm
Rosettagami(DE3): mutations in trxB and gor + extra copies of rare tRNA’s
SHuffle T7 Express: mutations in trxB and gor + cytoplasmic copy of DsbC → less reducing cytoplasm + isomerization activity
Tuner: lacZY deletion mutants of BL21. The lac permease (lacY) mutation allows a more uniform entry of IPTG into all cells, which makes a more concentration-dependent induction possible.
Lemo2(DE3): the T7 lysozyme level can be modulated by L-rhamnose
ArcticExpress(DE3): expressesthe cold-adapted chaperonins Cpn10/Cpn60. These are similar to GroEL-GroES, but active at 4-12ºC, which makes this a very suitable strain for expressing proteins at low temperatures.
LOBSTR-DE3: possesses modified copies of ArnA and SlyD, which have a lower affinity for Ni2+ and Co2+ resin than their wild type counterparts
NiCo21(DE3): possesses a modified copy of GlmS and CBD-tagged versions of ArnA, SlyD and Can
Miroux B. and Walker J.E. (1996) Over-production of Proteins in Escherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels J. Mol. Biol. 260: 289-298