failing to get expression of my recombinant protein with BL21 cells

7 posts / 0 new
Last post
vanila
vanila's picture
failing to get expression of my recombinant protein with BL21 cells

I have introduced my insert in CT TOPO vector and transformed competent BL21 cells with the same but failing to express the protein which is of 15 KD in this system at 1mM IPTG conc. please suggest me what to do.

20101975
20101975's picture
Please chek the compatibility

Please chek the compatibility of this insert into the vector & BV21 cells. As the compatibility test shod be done as trouble shooting.

RLS
RLS's picture
 Often, when I have

 Often, when I have difficulty expressing protein from a certain vector, I clone it into other expression vectors (pET20b, pET15b, pMAL, pBAD, etc.) SOme of these vectors use different bacterial cells for expression (TOP10, Rosetta, BL21 codon +). You could even try to express your protein in BL21 codon + if the system is compatible. It has tRNAs for rare codons. Also, you can vary the IPTG concentration and the time of overexpression. Usually, though, if you can't get these types of things to work, clone into a different vector. It's a quite small protein so make sure you are using something like a gradient gel, say 4-20% to make sure you are able to see the small protein. 

It's really not uncommon to have difficulties expressing proteins from certain vectors or strains of bacteria. Trial and error is your best shot if it's necessary for your work. If you have any more specific questions about the systems I've listed, please ask. Is your protein by chance insoluble (have you run out your pellet from your purification on a gel)? When I have found certain proteins that are toxic to E. coli, sometimes using the pMAL vector, which puts an N-terminal Maltose Binding Protein onto your protein (which can be removed or not), has been successful for me. It is actually expressed in TOP10 cells or LMG194; I actually had protein that I could only get expressing in LMG194 with pMAL after trying many different systems and combinations. 

Hope this helps and good luck!

RLS

vanila
vanila's picture
thanks for your suggestion

thanks for your suggestion but still i have a a problem that i doubt that my protein is probably going in inclusion form and that is why i fail to see it on gel. can you please suggest if how the get it out of it.
thanks.

Sami Tuomivaara
Sami Tuomivaara's picture
vanila,

vanila,

Here's something to look at from Current Protocols... Another one. You can find more from Google Scholar or PubMed.

Cheers,

mikan
mikan's picture
I have different problem with

I have different problem with vanilla.
my protein is not expressed in TOP10 but in BL21. I still search the reason. Can you help me to find the answer? Thank you

The FFM
The FFM's picture
Your vector probably doesn't

Your vector probably doesn't have the appropriate promoters to express your protein in TOP 10 cells  but it is OK for BL21

here is a table of appropriate vectors to express you protein if interest in different E.Coli strains.

Expression vector
appropriate host strain

pBAD vectors
Top10, >LMG194

pET vectors
BL21 (DE3)

pGEX vectors
BL21

pMal vectors
BL21, TB1

pProEx vectors
BL21, DH10B

pQE vectors
M15 or M15 [pREP4]

pRSET vectors
BL21 (DE3) pLysS

pTrcHis
BL21