Tag Info

Hot answers tagged

9

One important aspect when expressing a protein from a different organism in E. coli is that the codon usage of the original organism is likely different from the codon usage of the E. coli used for expression (Codon usage bias). Though the genetic code is degenerate, not all codons are equal. They might encode for the same amino acid, but organisms tend to ...


6

Have you tried putting your two genes on two separate plasmids (with different origins of replication and antibiotic selection, of course) and co-expressing that way? If the first of the two subunits is expressing well and the second isn't, that's probably because the ribosomes are falling off the mRNA before fully transcribing the second gene. Are the genes ...


4

A quick search on T7 cysteines gave some clues: Bacteriophage T7-induced DNA polymerase is composed of a 1: 1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Preparation of active subunits in the absence of sulfhydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active ...


3

Amazingly, yes. See High level gene expression in mammalian cells by a nuclear T7-phage RNA polymerase. The authors recognize that the T7 RNA polymerase tends to work only in the cytoplasm thus is unable to transcribe genes in the nucleus. To remedy this limitation, the T7 polymerase was fused to a nuclear location signal peptide to localize the polymerase ...


2

I found a very nice paper: Designing Genes for Successful Protein Expression, which covers most factors that determine protein expression. I post parts of it, because I am sure it will be useful to some of you. Translation can be controlled at the level of initiation and elongation. Initiation of translation is primarily dependent on the sequence of the ...


2

Mad Scientist covered potential codon-bias issues (which can be ameliorated with Rosettas), but more generally, I've seen tremendous variability in expression rates between different vectors (pET-28 vs pET-24) without any apparent reason. Our lab has had tremendous success with IPTG-inducible vectors (going from pBC-SK to pET-24 increased expression ...



Only top voted, non community-wiki answers of a minimum length are eligible