I'm going to express acylases in E. coli. How much mM of IPTG should I use for my plasmid? I've seen from 0,1 mM to 1,0 mM in literature, and it has been reported that my protein form "inclusion bodies" very easily. Anyone has experience and cold give some advices?
Thank you
Initially try with 0.1 mM IPTG and if you see all the protein in inclusion bodies, then you can increase the IPTG to 0.5 to 1 mM IPTG, where you can expect large amounts of protein in inclusion bodies from 1 liter culture.
A quick way to determine is by doing induction study of 5 mL culture with 0.01, 0.05, 0.1, 0.2, 0.5, 1.0 mM IPTG and doing whole cell processing and comparison on SDS-PAGE. This can be easily done in a single experiment. For two of my genes cloned in pET28a, I have found 0.05 mM IPTG conc. to be optimum for maximum soluble protein expression. So you need to find out.
See these two papers with highly overexpressed protein at 0.05 mM IPTG conc.
https://link.springer.com/article/10.1007%2Fs11274-017-2248-z
http://www.sciencedirect.com/science/article/pii/S1046592814002010?via%3Dihub
Hi there,
Are you looking for soluble acylase or acylase in inclusion bodies? If the former, you have to slow down protein production either by using low IPTG and/or low temperature during induction (15°C). If the latter, high IPTG and 37°C would be perfect.
There is an other way to slow down protein production: autoinduction medium which allow a smooth transition of usable carbon source from glucose to lactose via glycerol.
Your question is not clear? Do you want to express your protein as inclusion bodies or as soluble proteins? Induction of protein expression with low IPTG concentration at low temperature often results in expression of soluble proteins. However, many factors can contribute to expression of proteins in their soluble forms in E. coli. Thus, you need to optimize expression conditions for expression of soluble proteins. You can refer the paper on optimization of conditions for high-level expression of soluble proteins in E.coli. Here is the link to access the paper.
https://www.researchgate.net/publication/5603846_High-level_expression_of_soluble_viral_structural_protein_in_Escherichia_coli
Article High-level expression of soluble viral structural protein in...
Hi
try autoinduction method,it's very convenient and you do not have to watch cell density. There are papers for high density autoinduction too, where you can achieve high yields of protein but its a little more work
good luck
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