I cloned my gene with kozak sequences in pRetTRE3G but it does not express. I could see by qPCR that there is a transcript. The sequence was taken from NCBI and synthesize at IDT starting from ATG to TGA (start and stop codons) with kozak seq and restriction sites.
Can some one pour light on it to make it successful expression of my gene.
Hi Ram,
If obstruction occurs after transcription, either you need to codon-optimize your ORF, or the your expressionsystem (including the post-translational modifications) render the protein misfolded.
Did you include any tags to Western-blot cross check the expression?
Is this a heterologous expression system (i.e., the origin organism differs from the expression system one)?
Best of luck, and keep us posted!
Hi Marcin,
Thanks for your response. The sequence is already human from NCBI. Yes my vector pRetro-x- TRE3G is a expression vector with all necessary requirements. I checked another gene, that gives good expression. So vector seems ok. Yes i did include tag but it doesn't work. It was before the stop codon. I belive this is not heterologous system.
I am planning to test the tag at the START codon. any suggestion.
Have you sequenced your cDNA? Just because it was gene synthesized doesn't mean it is error-free. How large is the cDNA? It is not uncommon to see problems with expression of cDNAs 2kb or larger in retroviruses, esp. in the cases of membrane proteins (not sure if this is applicable to your GOI).
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression