I am using Arabidopsis thaliana gene (4-coumaroyl-coa ligase 3) At4CL3 to express through E,coli strain BL21 (De3) strain. I used Pet28a expression vector to express the protein with 6xhis tag. I checked its expression level, but there is no any sign of expression. I am sure, the Bacterial strain contain the At4CL3 gene without any mutation. Do i need to do any codon optimization? Has anyone expressed this protein using E.coli (Bl21 strain).
Could you please share any information along with REFERENCE Papers related to my problem?
I have added IPTG to induce expression overnight at 16 degree Celsius ( i try to induced expression at 20, 25, 30 degree Celsius too, but no sign of expression).
Yeah i sequenced the vector to confirm my insert. Everything is perfect. But no sign of expression.
I've had similar problem with my protein and we resolved it using autoinduction medium instead of IPTG. The publication of Fox and Blommel, 2009 : "Autoinduction of protein expression" might help you.
Clone GFP in the same vector as a control. It is easy to check whether the expression is induced and is satisfactory. If this does work, the system is O.K. and then You have to think about codon optimization. Of course if you see several arginine AGA codons - this can be the reason of low or very low protein synthesis. Use double stop codon ...etc. So there are many possible reasons of expression failure, but the easiest and the cheapest way to find them is to be absolutely sure about the system first.
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