I have cloned my gene at N-terminal region of pHIS2 vector. I have done double restriction digestion of plasmid and observed my target gene on agarose gel electrophoresis. Then I did the transformation of the plasmid into BL21. I have prepared the soluble and pellet fractions and analyzed on SDS-PAGE. I did not see the protein expression either soluble or pellet fractions. The same gene I have cloned in pET 28a vector and protein over-expressed in BL21. Due to some protein conformation problem, I am strict to change the vector (pHIS2) for cloning.
Did I get that right, your protein could be expressed when using the pET28a construct but not when using pHIS2? Are you sure about the reading frame of your ORF after subcloning?
Yes. I have checked ORF. I have used my previous primer for primer designing. Changed only the restriction enzymes. (pET 28a: Xho I & Nde I) (pHIS2: Nco I & Xho I).
I just checked and if I am not mistaken, pHIS2 is a yeast expression vector and does not have a T7 promotor (see http://www.addgene.org/vector-database/3038/). So this vector cannot be used for protein expression in E.coli with or without IPTG induction.
Conformation (is that what you mean?...not confirmation) of bacterially expressed recombinant protein can be affected by temperature. Try growing your bacteria at 30 or 34 degrees instead of 37.
Hi,
pHIS2 is a reporter vector that can be used in yeast one-hybrid assays to identify and characterize DNA-binding proteins, and in my own experience, the expression of the protein cloned is low. If you want to detect it, you must use a western blott. If your objective is express and purify the protein in yeast, you should use another vector like pYES.
I hope that this information is useful
It’s quite possible that there are mutations in the promoter of the construct or definitely generation of a new stop codon within the sequence. So I recommend you check you sequence at first. If your sequence is OK, then you can try different expression vetors and host cells. The following link may help you:
https://www.researchgate.net/post/A_pET28a_bearing_an_insert_is_transformed_into_E_coli_BL21DE3_But_the_protein_is_not_expressing_with_conc_of_IPTG_and_different_temperature
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