I’ve been stuck with a stubborn protein that refuses to express properly in E. coli. I’ve tried adjusting temperature and media, but no luck. Has anyone used an expression service for difficult proteins? Did it actually work, or was it just expensive trial and error?
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Oh, I feel this one deeply. I once had a protein that just refused to express — we tried everything from lowering induction temperature to switching promoters. Eventually, we realized the gene was too GC-rich and simply wasn’t playing nice with the host system. We ended up codon-optimizing it, and boom, it finally worked. So sometimes the issue isn’t the expression conditions but the gene design itself. Keep that in mind before you lose your sanity over low yields.