New BL21-Gold cells accelerate the protein expression process
Melody Jerpseth Bruce Jerpseth Lisa Breister Alan
Stratagene has created variants of BL21 competent cells, the new BL21-Gold series. Epicurian Coli BL21-Gold * competent cells lack the EndA1 nuclease and offer 100-fold higher transformation efficiencies than the parental strains. These two features eliminate the process of first cloning and sequencing many protein expression constructs in an EndA strain and then retransforming the plasmids of interest into the BL21 strains for expression.
Epicurian Coli BL21 competent cells are the E. coli strains chosen for protein expression when a gene of interest is to be expressed from the T7 promoter.## These strains have been engineered either to contain an inducible T7 RNA polymerase gene or to readily allow one to be introduced by phage infection.1 BL21 competent cells contain a mutation in the ompT protease gene, which is implicated in the degradation of some recombinant proteins. BL21 cells are derivatives of E. coli B and naturally lack the Lon protease, which may affect the stability of some recombinant proteins.2
Although the BL21 cell series is engineered for protein expression, these strains have two significant problems that limit their use as primary transformation hosts. Because BL21 cells are derived from the E. coli B cell line rather than the E. coli K-12 cell line, their efficiency is relatively low (1 x 106 cfu/g of pUC18 DNA). The other problem causing BL21 competent cell lines to be inadequate for initial transformation experiments is that they contain the EndA gene, which encodes an endonuclease that rapidly degrades miniprep DNA. This degradation makes restriction mapping and DNA sequencing (procedures usually performed to confirm that the protein expression construct is correct) from EndA+ cells very difficult. To retrieve and confirm the plasmid before performing protein expression studies, investigators first transform the ligation mix of the protein expression construct into a high-efficiency, EndA cell line, such as XL1-Blue competent cells. Then, a second transformation into BL21 cells is performed for protein expression.
Stratagene has introduced the Hte phenotype present in our highest efficiency strain, XL10-Gold ultracompetent cells3,, into the BL21-Gold competent cell series to eliminate the process of performing two transformation procedures for many expression constructs.The transformation efficiency of the BL21-Gold competent cell series is improved 100-fold, to greater than 1 x 108 cfu/g of pUC18 DNA. In addition, BL21-Gold cells are EndA , making recovery of plasmid miniprep DNA for sequencing and restriction analysis easier than in an EndA+ strain. We have isolated DNA from BL21-Gold cells using both alkaline lysis (see Figure 1) and boiling lysis miniprep procedures; the purified miniprep DNA was of sufficient quality and quantity to perform restriction analysis and DNA sequencing. BL21-Gold, BL21-Gold(DE3) and BL21-Gold(DE3)pLysS competent cells can be purchased separately for use with any T7-based expression system, such as Stratagenes Affinity protein expression and purification system.
In order to determine whethe r the genetic manipulations that created BL21-Gold cells affected their ability to overexpress recombinant proteins, a clone that encoded the c-Jun N-terminal kinase (JNK) protein was transformed into both parental BL21(DE3) cells and new BL21-Gold(DE3) cells. After induction of the chromosomal T7 RNA polymerase gene with IPTG, the cell extracts were run on polyacrylamide gels and stained with Coomassie brilliant blue dye. For the JNK protein, the amount of protein expressed from both strains of competent cells was indistinguishable. Expression of JNK is shown in Figure 2.
The BL21-Gold series of competent cells allows researchers to directly transform many protein expression constructs into an expression strain without having to do a second transformation. With an EndA genotype and a transformation efficiency that exceeds the parental strain, BL21-Gold competent cells accelerate the overall process of cloning a gene and expressing and/or purifying a recombinant protein.
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* Patent pending
U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending.