I cloned a fungal transcription factor gene, amplified from cDNA and inserted in pET28a. check the ORF by sequencing, now I have a problem in protein expression. Also observed very slow growth of Transformed PlysS cells. Can anyone help me?
Dear Arvind
I think your insert is highly toxic to the host cells, you please use a different host system.
Hi there,
As your target seems toxic to E. coli, you can consider in vitro expression using your plasmid together with T7 RNA polymerase and S30 extract. If you can afford the Roche RTS in vitro expression kit, go for it.
1) Did you check the protein expression on SDS-PAGE at time zero (without IPTG), then cell sample at time of induction (IPTG addition), then cell sample after several hours of induction on SDS-PAGE?
2) Also please check in your sequencing data if your gene of interest is in the right frame.
3)If your protein is in frame, correct orientation and toxic for the bacteria, you will see that transformed strain should grow slower than the empty vector transformed strain (liquid culture) and that will also say you that your expression is leaky. IF the protein is toxic you should switch to a better controled expression heterologous system (maybe BL21)
Use BL21(DE3)pLysS E. coli cell strain as T7 Lysozyme encoded in a pLysS plasmid that reduces the basal level of T7 RNA polymerase expression. T7 expression method is a system used for high-level protein expression and some amounts of basal expression of protein will take place in uninduced cells.
Also, I would suggest you to check if the protein is soluble or not.Insoluble proteins have a better expression at low temperatures. Also check the quality of IPTG.
Hope all of the above is helpful in resolving your problem.
Dear Hardik, I have check the insert by sequencing this is in frame all is ok. growth of transformed cells is very slow in comparison to untransformed BL21de3(PlysS). Expression is always checked by induced and uninduced parallel. i have also check the expression at lower temperature (16oC- overnight)
Dear Arvind, Another alternative to BL21(DE3) is to use a promoter that is less leaky than the T7 promoter. We have had good success with the rhamnose promoter, which can be used in BL21 or other types of E. coli, such as DH10B. The promoter is induced with L-rhamnose, and can be tightly repressed by glucose.
Hello Arvind, A rhamnose-inducible vector is available from Lucigen (look under the Expresso cloning section). Feel free to email me: techserv at lucigen dot com for more information.
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