no protein expression - (Oct/30/2007 )
hi all,
i am trying to express 4 mycobacterial genes in BL:21DE3 lys, four of them got expressed (in SDS-PAGE), while one is not expressing at all,this gene is the shortest,288bp coming to about 15KDa, as this gene was put in pET32a from which trx tag was removed.
this gene was actually taken as a positive control,and has been cloned and expressed in several genes,
IPTG is given in 1mM concentration, and cell pellet as well as media was taken at 0hr, 2hr, 4hr, and overnight at 4C and 28C.
Thank you in advance for any help,
please respond
You may try to incubate your bacteria in 37 oC to reach the OD then induce them with 0.1 mM IPTG at 30 oC. This might help to induce them. The other thing is to re-transform your bacteria and take a fresh colony and culture it following expression. I hope these help.
i am trying to express 4 mycobacterial genes in BL:21DE3 lys, four of them got expressed (in SDS-PAGE), while one is not expressing at all,this gene is the shortest,288bp coming to about 15KDa, as this gene was put in pET32a from which trx tag was removed.
this gene was actually taken as a positive control,and has been cloned and expressed in several genes,
IPTG is given in 1mM concentration, and cell pellet as well as media was taken at 0hr, 2hr, 4hr, and overnight at 4C and 28C.
Thank you in advance for any help,
please respond
Did anyone able to express the one you can't? I think it might be the protein half life?