I am using pTD-NStrep-His vector. I have cloned two genes in two individual vectors. Those genes are two cellulase enzymes. I used Gibson kit during the cloning process and all results were great starting from amplification of gene to cloning into E.coli BL21 (DE3).
I have added both His-tagX6 and Stop codon (TAA) to the reverse primers so i don't have to add extra amino acids to reach the histag from the backbone vector.
The total concentration of Streptomycin (25ug/ml).
The expression was carried out at 37C until OD 0.7, Induction with IPTG was at final concentration of 400 µM. Samples were incubated at 25C for overnight. Then cells were harvested and extracted using 1% Triton X-100 + 1 M urea.
By checking on SDS-PAGE, i could not detect any of the proteins in both cultures. Those are the SDS-PAGE of the purified histag and the elution fractions for both samples.
The enzymes MW are 35 and 44 kDa.
Histag1: the first enzyme fraction bonded to histag
Elution1: the eluted fraction (un-bonded) fraction
and so on..
As i ve gone through before, this enzyme has been produced by few researchers without using signal peptide. Thus, enzyme will be active by extracting from the E.coli cells.
The frame was sequenced and ligated in the proper frame.
However, I am wondering if i am facing one of these problems.
1- The vector pTD-NStrepHis is an expression vector which works by restriction enzymes as mentioned in Addgene database (supplier).
https://www.addgene.org/45936/
Is it ok to use Gibson assembly kit in this case ?
2- The enzyme could be produced as inclusion bodies and precipitated? If so, how can i analyze/extract the inclusion bodies for SDS-PAGE?
Any advise? or other problems i might have ?
Hi there,
There is no simple answer to your question... To have a clear picture of the situation, you should compare SDS/PAGE patterns of samples obtained with the same amount of non transformed cells, non induced transformed cells and induced transformed cells. It's hard to state on expression/no expression if you don't have any referential to rely on (especially if the expression level is quite low). In a second move you would have to state on the solubility of the protein expressed by comparing SDS/PAGE patterns of total crude lysate and supernatant of centrifuged crude lysate. If your proteins are expressed in inclusion bodies you should spot them in samples from total lysates or cells and get nothing in lysate supernatant.
Source of cellulase enzymes ? Does they have CBMs, disulphide bonds and they require any glycolsylations ? Please check them before you clone and express in E. coli.
Already checked. They both are produced by Bacillus and recent work has done cloning in similar E.coli expression cell.
reviews the codon bias ??? if you have more than five rare codons you get a bad expression.
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