ArcticExpress Competent Cells are engineered to address the common bacterial gene expression hurdle of protein insolubility. These cells are derived from the high-performance Stratagene BL21-Gold competent cells, enabling efficient high-level expression of heterologous proteins in Escherichia coli.
Overcoming Protein Misfolding and Insolubility
Forced high-level expression of a heterologous protein in E. coli can result in the production of large amounts of incorrectly folded protein, generating aggregates of inactive protein known as inclusion bodies. While the aggregated protein may be easy to purify, obtaining active protein from inclusion bodies typically requires protein-specific and labor-intensive in vitro re-folding steps, with no guarantee of obtaining biologically active product. The ArcticExpress series of competent cells provides an in vivo approach to increasing the yield of soluble protein produced in E. coli.
The mesophilic host E. coli is suitable for expression of a wide range of heterologous proteins. At standard cultivation temperatures, however, high-level expression of a heterologous protein can impair the cell’s ability to properly process the recombinant protein. Low-temperature cultivation represents one strategy for increasing the recovery of soluble protein. An obstacle to using this approach, however, is that E. coli chaperonins, which facilitate proper protein folding by binding to and stabilizing unfolded or partially folded proteins, lose activity at reduced temperatures. Specifically, it has been shown that the activity of the E. coli chaperonin complex GroEL/ES retains only about 30% refolding activity at 12°C, compared to its activity at the temperature optimum of 30°C. To overcome this obstacle, ArcticExpress competent cells have been engineered for improved protein processing at low temperatures. These cells co-express the cold-adapted chaperonins Cpn10 and Cpn60 from the psychrophilic bacterium, Oleispira antarctica. The Cpn10 and Cpn60 chaperonins from O. antarctica have 74% and 54% amino acid identity to the E. coli GroEL and GroES chaperonins, respectively, and show high protein refolding activities at temperatures of 4–12°C. When expressed in ArcticExpress cells, these chaperonins confer improved protein processing at lower temperatures, potentially increasing the yield of active, soluble recombinant protein.
Protein Expression Systems and Induction Methods
The ArcticExpress strains are designed for expression of recombinant proteins either from the T7 promoter using the DE3 lysogen strain, or from IPTG-inducible, non-T7 promoters using the non-DE3 lysogen strain.
Expression from the T7 Promoter For recombinant proteins expressed from vectors driven by the T7 promoter, such as the pCAL vectors and the pET vectors, use the DE3 lysogen ArcticExpress (DE3) host strain. This host strain contains a chromosomally integrated cassette in which the T7 RNA polymerase is expressed from the lacUV5 promoter. Induction of T7 RNA polymerase expression with IPTG results in expression of the T7 promoter-driven recombinant protein.
Expression from Other IPTG-Inducible Promoters For recombinant proteins expressed from IPTG-inducible promoters other than the T7 promoter, such as the tac or trc promoter, use the non-DE3 lysogen ArcticExpress host strain.
Overcoming Codon Bias
Efficient production of heterologous proteins in E. coli is frequently limited by the rarity of certain tRNAs that are abundant in the organisms from which the heterologous proteins are derived. High-level expression of heterologous proteins can deplete the pool of rare tRNAs and stall translation. ArcticExpress RIL and RP strains are engineered to contain extra copies of genes that encode the tRNAs that most frequently limit translation of heterologous proteins in E. coli. Availability of these limiting tRNAs allows high-level expression of many heterologous recombinant genes that are poorly expressed in conventional BL21 strains.
ArcticExpress RIL Strains ArcticExpress RIL and ArcticExpress (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 (Table I). The ArcticExpress RIL strains have available the tRNAs that most frequently restrict translation of heterologous proteins from organisms that have AT-rich genomes.
ArcticExpress RP Strains ArcticExpress RP and ArcticExpress (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 ArcticExpress RP strains have available the tRNAs that most frequently restrict translation of heterologous proteins of organisms that have GC-rich genomes.
For Research Use Only. Not for use in diagnostic procedures