Fail to expression recombinant protein

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Happy725
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Fail to expression recombinant protein

Dear all,
I have problem to express my recombinant protein, which will be used to raise Ab. I cloned a full ovine gene (954bp) with a 6his tag at 3' in frame into 2587bp pRUN (modified from pET) expression vector using C43(DE3) strain. DNA sequence was confirmed. A O/N culture was refreshed until OD 0.4, and IPTG added to finial 1mM (~4h). 1ml culture were tested before and after induction, SDS-PAGE showed the extract same patterns. The gene has 83%GC rich exon 1 sequence (83bp), is that could be a problem?? The protein has unknown function, and predict to be a toxic protein. What shall I do?? Thanks

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HI,

HI,

If you let me know the complete details of your experiement so that it will be easy to draw a conclusion or give suggestions on your experiement. Did you check your gene is sitting in the right orientaion and frame? Did you check  rare codons present in your gene or not ? did you try  tried different hosts and temparature , IPTG, Length of induction at low temp.

If your answer is yes for all / most of the questions then, some times you might not see overexpression in SDS-PAGE gels as expressions levels will be very low. So, check the expression by western blotting with anti his antibody etc.,

If your answer is no for all / most of the questions try to do  those experiments to draw a conclusion.

All the best.

Balu..

Happy725
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Morning Balu..

Morning Balu..
Thanks for your suggestions, I will check the expression by western blotting with anti-his Ab. But I did not get what do you mean by "rare codons present in my gene"? Does the GC rich exon 1 sequence may affect the protein expression? If I exclued that part to re-construct, is that might be working???
Here is what I did.......5’ (BamH I) and 3’ (Hind III) enzyme were used for construction, so DNA sequencing confirmed insert is in right orientation. But one thing is odd for the cloning. The sequencing of the insert PCR is perfert, but alwarys got several nucleotides changes on my inserts, some of them did not change aa, some do. I do not know this happened? I tried cloning for several times, a couple of aa always changing, but in different random places. I used site direct mutagenesis kit to change the normal aa of my insert back, and transform the correct plasmid DNA into different competent cells (DH5a/ BL21/ C43(DE3)/JM109), grow coloines on agar plates with Amp(100μg/ml) as the selective antibiotic.

A single colony was grown in 10 ml LB with Amp at 37°C O/N, 250 rpm. 1ml of the O/N culture was re-freshed with 50ml LB with Amp, shaking with the same condition till OD=0.4. 1ml sample was taken out, and spin down for pellet. IPTG was then added, I tried different finial conc. (1, 0.5, 0.2, 0.1mM), and different induction time (30min, 1h, 2h, 4h, 6h, O/N). 1ml samples were taken out after induction. I also tried different temperatures (25°C and 30°C). All the sample from 1ml culture were respended using 100ul PBS, 10ul was mixed with 5ul protein loading dye, boiling for 10min, spin down and checked with 9% SDS-PAGE gel along with marker.  All the gel showed the similar bands in same pattern. I have been working with other proteins, if expression works, should be visible very obvious.