12 replies to this topic

[alantkl]

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.

[protolder]

alantkl, on 15 March 2011 - 04:42 PM, 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 don´t see any signal in Western-blot I´ll recheck the sequence and frame. Give us more details, Buena suerte

[Rutenium]

Have you tested expression at different temperatures (+37C and RT, for example)? Also, what strain and vector are you using?

[HomeBrew]

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.

[alantkl]

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.

[pDNA]

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 16 March 2011 - 04:41 PM, 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.

[alantkl]

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.

[protolder]

alantkl, on 17 March 2011 - 10:37 PM, 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 don´t purify anything, you have to revise the cloning again. Buena suerte

[alantkl]

Anyone has experience using Novagen pTriEx system?

[chimaera]

IMO the autoinduction protocol (uses slow lactose induction instead of IPTG) as described here: http://www.microbiol...Induction.shtml can sometimes work for difficult proteins.

[alantkl]

Thanks chimaera. Just wondering the recombinant plasmid should be isolated from the cloning host and directly transfect into the expression host for the Novagen pTriEx system, am I right?

[protolder]

alantkl, on 31 March 2011 - 05:59 PM, said:

Thanks chimaera. Just wondering the recombinant plasmid should be isolated from the cloning host and directly transfect into the expression host for the Novagen pTriEx system, am I right?

Hola,Yes with the plasmid from cloning strain you can tranfect mammalian cells or insect cells , in this last case for the homologous recombination method with Bsu36I digested baculoviral DNA from Clontech, Orbigen,  Pharmingen or bacvector Novagen etc.This plasmid isn´t compatible with Invitrogen systems Bac-to-Bac, Baculo direct, and blue bac buena suerte con la expresión

[alantkl]

Gracias amigo