I am trying to express sheep Tyrosinase protein with wild type and mutant type in E. coli bacteria. I have cloned my genes in pGEX-6P-1 vector. I have confirmed the cloning of gene by sequencing. Now i am doing expression in BL21 (DE3) bacterial cells at 37 C temperature. I induced protein by 0.6mM, 0.8mM and 1mM IPTG at stage 0.8/0.6 OD600 at 37 C, 30C, and 20C temperature. After IPTG induction i collected samples at 2 hours, 5 hours, 8 hours and 15 hours interval. I confirmed the protein expression by running those collected samples on SDS-PAGE. But i could not get the protein expression. I did this experiment lot of time but could not get the protein. I am much worried about this. There is something wrong with my method.
I need your precious guidance and experience in this regard.
Thank you
The first thing I would try is to retransform your plasmid into fresh BL21(DE3) cells. Sometimes your existing transformant has accumulated a mutation that stops expression. If that still does not work, then I would use some other known control plasmid to make sure your conditions and media are good and your BL21 strain is good.
How's Rafiz doing, all right ?! Work with molecular analyzes with human blood (qPCR). I do not know how to carry out your protein analysis, but I suggest you talk to Dr. Almir de Souza Martins (Biologist who specializes in all types of molecular biology analysis). Good luck!!
Hi Hafiz,
Choice of growth conditions looks okay.
BL21 DE3 is quite a leaky strain which carries phage T7 RNA polymease that only recognise T7 or T7-lac promoters while not use BL21 that lacks this phage polymerase but recognises the tac promoter by its own RNA polymerase for your pGEX-6P-1 vector to expression your gene?
Do check your insert sequence is in frame to translate the Sheep Tryosinase correctly?
collect uninduced samples at the time interval? Helps with comparison with induced samples even if you got no band on SDS PAGE
Not sure the Sheep Tryosinase is a soluble protein but do analyse the pellets of your lysates? Maybe its part of inclusion bodies and best incubate at lower temperature.
If that not working maybe you should use strain containing the rare codons of Eukaryotic tRNAs with T7 lysoyzme (either in strain or on vector) e.g. Rosetta Gami plysRARE (find a list on NOVAGEN) and clone into a pET vector (BL21 DE3 is suitable for pET vector)
Hope this helps. Goodluck
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