I have already cloned ALP gene of L rhamnosus into pET22b+ vector. Cloning is already confirmed by restriction digestion and sequencing. Cloning is achieved in BL21(DE3), Codon+, Rosetta and Lemo cells. Different concentration of IPTG has been used starting from 0.1mM to 1M. Different temperatures like 37oC 18oC 16oC were used to achieve expression.Still there is no expression. Under simulation I have used Snapgene to construct vector and simulate expression. Everything was perfect but still there was no expression after Induction and SDS PAGE. Please do suggest.
Did you clone the mature (not the entire) coding region in-frame with the PelB leader?
Sir I have cloned the entire region of gene (PCR purified) in frame with PelB leader. Moreover the CAI score for gene was 0.66. Is that a problem?
Have you sequenced your final construct to be sure everything is correct?
When you say there is no expression, do you mean there is no activity or no protein? Can you see protein by western blot? You need to distinguish between whether the enzyme is not active in E. coli or whether it truly is not expressed.
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