I need some proteins (S100es) to standardize my ELISA. I want to express and purify it by itself in E.Coli. However, I am confused about vector plasmids, which plasmid will be good for high yield, and which media I should use. I also think choosing a proper E.coli strain is very important. Can anybody suggest a protocol, some literature, or some general advice?
The expression yield is depend on many matters, such as rare coden, molecular weight, posttranslational modification, toxicity, etc. You need to tell as in detail.
generally, BL21(DE3) with pET series are pretty good for expression. Also pCOLD series are good in soluble expression, however low inducing temperature is required.
I want to express these proteins; S100A8, S100A9, S100A12 und S100A14. These are small Ca ion binding proteins. I have idea about posttranslational modifications, as there lenght is about 104-110 amino acids and these are Homodimers and each monomer consists of 2 Ca-ion EF-hand domains.
No they are not toxic.
Small and acidic proteins. Size 10-12 kD.
I dont have any idea about posttranslation modification; I am going to search, but I dont think that there is some special modification after translation.
Actually I am not good in this field.
I found some modifications, are these important or not?
Proteins Modifications
S100A8 phosphorylation
S100A9 phosphorylation und methylation
S100A8/S100A9 heterodimer (FA-p34) complex binding of unsaturated fatty acids with high affinity
S100A12 S-sulfo-cysteine
Aaqib
Hi. We used rosetta gammi strain of Ecoli and PET (pet32a) system for expression of several eukaryotic proteins. As Sacha mentioned , this strain doesnt need to codon optimization and has high level expression and has a good yeild.
Good luck
Aaqib
Hi. We used rosetta gammi strain of Ecoli and PET (pet32a) system for expression of several eukaryotic proteins. As Sacha mentioned , this strain doesnt need to codon optimization and has high level expression and has a good yeild.
Good luck
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