Dramatic induction was observed, but no desired protein band was observed. I run an empty pET as a control. There doesnt seem to be difference with the empty pET and pET with insert. I have no time to go for western blot as purchase of antibodies for western will take more than three weeks :( any suggestions?
Hi, have you done RE analysis to check whether your target insert is cloned on pET vectr or not..? is insert release is there on RE analysis, go for sequncing to verify that the your gene of interest is in proper reading frame or not?
If the size of your protein is bigger then there is a chance that it wont get induced.
what is the size of your protein of interest.Also do the restriction analysis of the clone for confirmation of gene of interest.
Lack of insert and incorrect reading frame are both well known and key issues to check before anything else. Other issues that can lower the level of expression of exogenous proteins in bacteria include (1) species of your protein of interest. Codon bias can be a problem. (2) Try IPTG vs autoinduction, I have seen it give radically different levels of expression. No way to predict which works best for your protein, try and compare. (3) Some proteins just don't express well in bacteria. Could be missing an obligate binding partner so unstable or other issues. Try other expression system.
In what condition do you induce bacteria? ( I mean concentration of IPTG, OD of induction, duration of expression)
If you just want more expression, the bacteria induced by IPTG should be incubated for long time( 12h or 24 h) in specified tempreture. ( but long time is n't good for some protien like enzyme)
If you have doubt about the presence of desired gene in pET, do cracking.
I have already verified he clone via RE analysis and sequencing. I have converted the DNA seq to amino acid and then align accordingly. I am positivie that it is in the correact reading frame. I am trying to express a lipase gene from rice. I am using Rosetta in hope to overcome the problem of codon bias. I am inducing the bacteria at 37C & 16C overnight at OD of 0.9, with 1mM IPTG concentration.
Dear Shanice,
Could you provide more details about your experiment? Like the protocol you followed, size of the overexpressed protein and a gel picture perhaps?
Thank you all for the suggestion! I have also tried the BL-21 (DE3). Both Rosetta and BL21 do not seem to express the protein. I transform the recombinant plasmid to the Rosetta or BL21 each time. I didn't plate the cell on the plate but instead I use the cell culture right after recovery to innoculate (with antibiotic). I then grow it overnight at 37C. On the next day, I subculture it in a fresh broth and grow till the OD reaches ~0.9. Following that, i then tried to induce with 1mM at two different temperature 37C and 16C. The size of my expressed protein should be around ~25kDa.
Dear Shanice,
If you grow and induce directly from your recovered culture after transformation, you will select for bacteria which are growing fast, like those which don't express your implanted protein, unless the latter gives them an advantage in propagation (which usually is very unlikely).
Thanks dr Wolfgang. I happened to read some sources that say it's not necessary to plate on plate. But well your comment make so much sense. I will try to transform again and plate it tmrw. And try with protein expression again. Thanks for pointing out this prob
I think you should check out your DNA sequence cloned. Sometimes, we think the correct DNA sequence for protein is cloned, but sometimes a little error changes the lecture frame. It happened once tom me. Have u sequenced your vector? Commercial vectors usually includes the primers for sequencing on MCS.
Dear Shanice,
I had similar problem before. It would be worthy to change the competent cell, culture media and induction condition. In my case, I solved the problem as following. I plated the reaction of transfomation (I guess it would be better to pick colony from the plate unless the gene is toxic to bacteria. I assume that T7 promoter vector like pET would not work very well if gene is toxic ). I thought that it is somewhat difficult to make the recombinant protein from bacteria. I changed the cell to BL21 (DE3) RIPL cultured with LB, not TB. I reasoned that lack of some tRNA is problem. However, I could not get high expression. So I cultured the cell at 25'C for ~24hours. The results was really good.
Good luck,
You can use zymography methods to check your protein activity. if it does not ok. i think you cut vector by restriction enzyme to ensure your gen in vector. if possible you sequenced your gen. it's the best plan.
everything is ok. you transform vector into cell. you can use other cell. in vietnam i use BL21 DE3, jm109... you can use ampiciline with more concentration. a tips, you incubate it less than 14 hours because when you incubate more than 14 hours it maybe have satellite colonies.
when expression you use LB, SB or TB. if you want protein solute you decrease temperature. my protein solute at 20'C and 25'C..
good luck, my english is not good. hopefully, you can understand my idea :)
Thanks Sung and Lam. Thank you so much for your valuable suggestions. I have already sent my plasmid for sequencing and after transformed into the expression vector BL21(DE3) or Rosetta (DE3) I did an insert check sing PCR. I will try them out and continue to pray for good results.
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