Protein expression in E.coli host strains

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msahar
msahar's picture
Protein expression in E.coli host strains

Dear all,
I am working on the recombinant protein expression in different host strains.I have expression of my protein but almost 90% of expressed protein is in the insoluble part of the cell lysate. I have played around the IPTG conc. and also different expression strains but yet no significant change.What to do?
Waiting

Sangram
Sangram's picture
Your protein of interest

Your protein of interest might be going inside inclusion bodies.

you may try with insoluble protein extraction buffer any standard company . For example...
http://www.genscript.com/product_001/kit/code/L00230/category/kit/Protein_Extraction_Kit_Bacteria.html

best of Luck!

RLS
RLS's picture
I've had great luck with the

I've had great luck with the pMAL system from New England Biolabs (http://www.neb.com/nebecomm/products/productE8000.asp). I've been using the pMALc2x vector for cytoplasmic expression in E. coli, but there are also vectors for periplasmic expression if the cyto expression doesn't work. It produces a fusion protein of your protein of interest preceded by maltose binding protein and a linker. You can subsequently cleave the MBP off. I've been able to get soluble expression and active protein for several things using this vector, whereas, every other vector and/or E. coli strain didn't help.

This is especially helpful if you are working on a sensitive enzyme and you want to retain activity by minimizing the number of steps for purification!

kjosephs
kjosephs's picture
Can you say what protein or

Can you say what protein or type protein you are trying to express in E. coli and is this something published that you are trying to reproduce or something new? Also is the 10% of the protein that is soluble properly folded, do you have an activity assay?

For some types of proteins if you are getting 10% soluble expression and that protein is active, then that's great, and it is usually enough to support a project, ie, you can get enough protein by scaling up.

Vectors that fuse various proteins (GST, MBP, Thirodoxin, etc), IPTG concentration, and host strains are all worth trying.

I would also try a lower temperature and extending the induction time. For example, expand the cells to about 0.1 OD600 at 37C, then shift to 22C for the last 3 doublings to OD600 0.6-0.8, then add IPTG and allow induction at 22C to proceed over night.

Tirumal Rao
Tirumal Rao's picture
hi

hi

The optimal IPTG condition and how many hours after to keep for shaking and then the temperature at which u grow also affects ur protein expression

it differs for different proteins in BL21 , only u can determine that amount or temperature or how many hours

so first optimize it
try a range from 0.1--1mM IPTG at 37 for 4-5 hours after induction
if ur lucky to get the optimal IPTG concentration,

then use that concentration for a variation in time for which u incbuate after induction from 1- 7 hours
generally IPTG induction is carried out for no more than 5 hours but it depends on your protein. sometimes, to reduce proteolysis/ degradation of the protein, you can induce at room temperature for about 3 hours, nice and easy. i've used both 3 hours at 25 degrees C, and 4 hours at 37 degrees C, the former purification was much cleaner in terms of degradation products; still, it's protein relative.

after that use that time and IPTG concentration to try at different temperatures like 20-25-30-37-40 etc

Tirumal

Rajeshwari patel
Rajeshwari patel's picture
hi,

hi,
 
I agreed upon tirumal answer one more thing you tried upon it slight basic condition in your induction media

asif123
asif123's picture
 Dear Tirumal,

 Dear Tirumal,
Could you please tell me the cell density or OD generally suitable for IPTG induction and few most effective media for E.Coli BL21.

Thanks
Asif

asif123
asif123's picture
Dear kjosephs,

Dear kjosephs,
In you answer "For example, expand the cells to about 0.1 OD600 at 37C, then shift to 22C for the last 3 doublings to OD600 0.6-0.8, then add IPTG and allow induction at 22C to proceed over night". it is not clear for me why you want to reduce the temp at about 0.1OD600?

Sincerely
Asif 

wyzandrea
wyzandrea's picture
Successful recombinant

Successful recombinant protein expression and purification is dependent on a robust and efficient proteomic workflow. From small-scale to large-scale studies, our reagents, kits, and vectors are designed to address all needs including induction of protein expression, cell lysis/extraction, and capture and purification. Discover how Sigma can enhance your gene regulation, structural, or functional assays today!
From Creative Animodel!

Successful recombinant protein expression and purification is dependent on a robust and efficient proteomic workflow. From small-scale to large-scale studies, our reagents, kits, and vectors are designed to address all needs including induction of protein expression, cell lysis/extraction, and capture and purification. Discover how Sigma can enhance your gene regulation, structural, or functional assays today! - See more at: http://www.sigmaaldrich.com/china-mainland/zh/life-science/proteomics/recombinant-protein-expression.html#sthash.IcZErlVM.dpuf
Successful recombinant protein expression and purification is dependent on a robust and efficient proteomic workflow. From small-scale to large-scale studies, our reagents, kits, and vectors are designed to address all needs including induction of protein expression, cell lysis/extraction, and capture and purification. Discover how Sigma can enhance your gene regulation, structural, or functional assays today! - See more at: http://www.sigmaaldrich.com/china-mainland/zh/life-science/proteomics/recombinant-protein-expression.html#sthash.IcZErlVM.dpuf
Successful recombinant protein expression and purification is dependent on a robust and efficient proteomic workflow. From small-scale to large-scale studies, our reagents, kits, and vectors are designed to address all needs including induction of protein expression, cell lysis/extraction, and capture and purification. Discover how Sigma can enhance your gene regulation, structural, or functional assays today! - See more at: http://www.sigmaaldrich.com/china-mainland/zh/life-science/proteomics/recombinant-protein-expression.html#sthash.IcZErlVM.dpuf
Successful recombinant protein expression and purification is dependent on a robust and efficient proteomic workflow. From small-scale to large-scale studies, our reagents, kits, and vectors are designed to address all needs including induction of protein expression, cell lysis/extraction, and capture and purification. Discover how Sigma can enhance your gene regulation, structural, or functional assays today! - See more at: http://www.sigmaaldrich.com/china-mainland/zh/life-science/proteomics/recombinant-protein-expression.html#sthash.IcZErlVM.dpuf

Mike Dike
Mike Dike's picture
protein expression

Hi,I have been trying to express a recombinant protein of  ~100kDa but on two different occasions the results from my expressions (SDS-PAGE) showed thicker bands in the samples before induction than after induction. Ordinarily, I would have expected, for a good expression of my target protein, to see thicker bands in the samples taken after induction than before - but the reverse was my observation.Please what could be wrong?