Hi there, if there is no protease cleavage site in the initial plasmid there is no chance to manage to get rid of the GST. You should clone your target in a convenient vector.

Is there a way to cleave a GST tagged proteins using pDEST15 from gateway system. I realised the pDEST didn’t have Protease cleavage site?

In my view you have succeeded to clone and express your gene of interest in a recombinant GST tagged protein using pDEST15 in BL21-Al

However you haven’t the option to cleave between the GST peptide and your protein to obtain a free recombinant protein.

I advise you to integrate one protease cleavage site at the N-terminal sequence of your protein, I mean you clone the nucleotide sequence in the 5’ end of your gene. Just before the ATG codon, you should be care that the cloning gives an open frame sequence ORF with your protein sequence.

Methodology, For Ex the Fac enterroxinase sequence: DDDDK/ the nucleotide sequencecould be : GAC GAC GAC GAC AAG

You can add the cleavage sequence by PCR using a 5’ Forword primer with the as 5’ GAC GAC GAC GAC AAG ATG (Met) NNN (Aa2) NNN(Aa3)…..

After that you could produce and synthesis your DNA sequence by a PCR protocol with the this 5’ Forword primer and the Reverse Primer of your gene sequence. The PCR product could be cloned pDEST15 in BL21-Al under the GST Tag, In all cases you should always verify the ORF: GST peptide-Protease tag-Protein of interest.

In parallel, I indicate you a set of 4 proteolytic tags with their specific protease

1) Enterrokinase cleavage: site DDDDK/ DNA sequence 5’: GAC GAC GAC GAC AAG

2) (Factor Xa) cleavage peptide: IEGR/ DNA sequence 5’ATC GAA GGT CGT

3) TEV (tobacco etch virus) protease: ENLYFQ/G

4) Thrombin cleavage site: LVPR/GS

I also advise you to use the enterokinase tag or Factor Xa tag since after proteolysis of the recombinant protein you obtain a free protein without any Amino acid addition. However, The others tags 3) and 4) will give an Amino acids addition G and GS respectively

Good work

With best reagards

Thank you @ Liger

Thank you so much Mohamed Nejib Marzouki

Mohamed Nejib Marzouki , if I include the cleavage sequence, before cloning I will need to include attL1 and attL2 sequences for cloning with pDEST15 because the cloning uses recombination. Im I right? Thank you

if I include the cleavage sequence, before cloning I will need to include attL1 and attL2 sequences for cloning with pDEST15 because the cloning uses recombination. Im I right?

Could you explain me how you have cloned your gene in pDEST15 bordered by the sequences attL1 and attL2?

However, If you have prepared your gene sequence (cDNA) by PCR, you may perhaps amplify it by the Forword Primer: ATG (Met) NNN (Aa2) NNN (Aa3) NNN (Aa4)….. and your commonly Reverse Primer, so you could as I explain previously, integrate the protease cleavage sequence before the ATG ‘Met), Yet, you may use this Forword primer : 5’protease cleavage sequence-ATG-NNN(Aa1)-NNN(Aa2)-NNN(Aa3)-…….after that you purify your PCR product that contain your gene located downstream the protease cleavage sequence and therefore you cloned it in the pDEST15.

I want to notice that you have to firstly verify that your Protein polypeptide is not hydrolyzed by the selected protease (I mean that your gene sequence doesn’t contain the proteolysis cleavage site) and secondly to verify always the ORF sequence to express correctly the amino acid sequences of both Protease cleavage peptide and Gene of interest protein.