In cases when toxicity of the gene of interest is an issue in these expression host strains, the use of hosts carrying the pLysS or pLysE plasmids may be beneficial. For recombinant protein production, we recommend transferring the vector to BL21(DE3) or HMS174(DE3) host bacteria strains, which carry chromosomal copies of the T7 RNA polymerase gene driven by the LacUV5 promoter. coli strain designed to maximize plasmid integrity and lacking the T7 RNA polymerase gene (such as Stbl3). Additionally, the system is able to maintain the gene of interest in a transcriptionally silent state when T7 RNA polymerase is not present.Īll custom pET vectors will be supplied in an E. This can be advantageous when expressing proteins with limited solubility. Addition of IPTG blocks the inhibitory action of LacI, thereby inducing expression of T7 RNA polymerase and also removing LacI inhibition of the gene of interest.Īlthough the pET expression system is designed for high-level recombinant protein expression, the expression level can be reduced by decreasing the amount of IPTG supplied to host cells. The LacI protein acts at the LacUV5 promoter in the host cells to repress expression of the T7 RNA polymerase gene by the host polymerase, and also functions at the T7lac promoter on the pET vector to block transcription of the gene of interest by any T7 RNA polymerase that may be made due to leaky expression. The plasmid also carries the natural promoter and coding sequence for LacI. Additionally, there is a lac operator (LacO) sequence just downstream of the T7 promoter that can be acted upon by the lac repressor (LacI) protein to block transcription of the T7 promoter. In this system, there is a T7 promoter that can be acted upon by T7 RNA polymerase to drive high-level expression of the gene of interest. The vector utilizes the T7lac promoter system for strong and tightly controlled gene expression. coli, which reduces leaky expression before induction. The pET vector exists as a low copy number plasmid in host E. Expression of the T7 polymerase is induced by the addition of the lactose analog IPTG to the bacterial culture. λCE6 phage), or more commonly, the pET plasmid can be transferred into a bacteria host strain whose genome has been engineered to carry the T7 RNA polymerase gene under the control of theLacUV5 promoter. The host cells can be infected with phage carrying the T7 RNA polymerase gene (e.g.
Afterwards, expression of the gene of interest can be initiated in two possible ways. This eliminates plasmid instability due to expression of proteins of interest that may be harmful to host cells. coli and insect cells.The gene of interest is initially cloned into the pET vector in a bacteria host that lacks the T7 RNA polymerase gene. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and a database system dedicated to handle co-expression data are described. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity.
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