improving recominant protein expression

[janu]

hi all

i have cloned 600 bp gene in pet vector

in colony pcr i have got positive result ,i have got amplification in that

once after expression analysis of protein with IPTG induction i am getting very less amount of expression

can anybody suggest me how to improve expression of target recombinant protein

thanks in advance

[ecoli]

without knowing anything more about your protein or strain, I would say try inducing for longer or maybe using a higher concentration of IPTG. Try a bunch of different conditions and run then on an SDS-PAGE gel simultaneously.

[janu]

protein is human growth hormone and strain is e coli bl21

[CharonY]

Unfortunately there are a gazillion possibilities why it does not work. First question is whether there is production of the correct protein at all. Based on the OP I'd assume that there was low production. Have you checked whether it is the correct protein (e.g. by MS/MS)?

[ecoli]

what vector? One time, I spent 6 months trying to successfully clone my insert into a pMMB expression vector.