Expressing human protein in E.Coli - (Mar/15/2011 )
I am having a hard time to express human protein in bacterial system. I have tried various concentration og IPTG, but it seems my protein is reluctant to be expressed. The incubation period tried out were 1h, 2h, 3h, 4h, and even overnight. I need assistance in this matter, Thanks.
alantkl on Wed Mar 16 00:42:03 2011 said:
I am having a hard time to express human protein in bacterial system. I have tried various concentration og IPTG, but it seems my protein is reluctant to be expressed. The incubation period tried out were 1h, 2h, 3h, 4h, and even overnight. I need assistance in this matter, Thanks.
Hola,Have you compared your culture with one with the empty plasmid?, depending of the host sometimes there is a constitutive expression without induce. Have you any antibody against your protein or tag?. If you donīt see any signal in Western-blot Iīll recheck the sequence and frame. Give us more details, Buena suerte
Have you tested expression at different temperatures (+37C and RT, for example)? Also, what strain and vector are you using?
You may need to transform your clone into one of the Rosetta strains that contain a pRARE plasmid. Check your gene's sequence for the number of codons it uses that are rarely used in E. coli.
Thanks for the prompt reply. I'm using pTriEx vector on Origami B strains (Novagen). Yes, I have compared between empty plasmid as negative, beta-galactosidase expression as positive, uninduced control and induced sample. But still not successful. Should I try on Western 'cos it's more sensitive in detection? I have my protein substrate with me.
western is definitly a good idea!
Have you discriminated between soluble and insoluble fraction ...maybe your protein goes strictly into inclusion bodies.
Regards,
p
alantkl on Thu Mar 17 00:41:23 2011 said:
Thanks for the prompt reply. I'm using pTriEx vector on Origami B strains (Novagen). Yes, I have compared between empty plasmid as negative, beta-galactosidase expression as positive, uninduced control and induced sample. But still not successful. Should I try on Western 'cos it's more sensitive in detection? I have my protein substrate with me.
I have extracted both soluble and insoluble cytoplasmic fractions, but I don see any difference on the gel. Probably, I should try out western. But Is there any alternative to detect the expression of my protein before western? I really hope to see it on my SDS-PAGE. Please advice.
alantkl on Fri Mar 18 06:37:06 2011 said:
I have extracted both soluble and insoluble cytoplasmic fractions, but I don see any difference on the gel. Probably, I should try out western. But Is there any alternative to detect the expression of my protein before western? I really hope to see it on my SDS-PAGE. Please advice.
Hola, but you have to see different patern of bands in each sol/insol fractions. Yes, you need see the band in direct SDS gel to try purify and have any significative amount. An option could be to put a bit (10ul) of the specific resin against tags let interact 10-20 min wash add loding buffer heat centrifuge and load the gel. if you isolate any band you coul think in made an scaling-up with more volume of sample and resin, but if you donīt purify anything, you have to revise the cloning again. Buena suerte
Anyone has experience using Novagen pTriEx system?
IMO the autoinduction protocol (uses slow lactose induction instead of IPTG) as described here: http://www.microbiology.emory.edu/altman/jdaWebSite_v3/p_tet_autoInduction.shtml can sometimes work for difficult proteins.
