I recently cloned an E. coli optimized gene for recombinant protein expression into a pET-based plasmid containing an N-terminal MBP fusion under T7 Promoter control, however while I am getting expression of the desired protein, it is less than I would have expected (~10-20% of protein after lysis).
Currently, I am inducing BL21(DE3) E. coli with 0.25 mM IPTG for 16 h at 18°C (conditions that were all optimized for this particular experiment) and purifying by NiNTA followed by ion exchange chromatography. I'm wondering if there are any other parameters I can optimize to boost my yield, or if it is worth switching to a different vector (such as the pMAL-c5X from NEB, which is under a different promoter)?
Dear Chris,
Depending the type of protein you're working with 10-20% of the total protein after lysis is actually quite a bit !
Have you tried different media for the culture ? There is for instance TB (terrific Broth) media or 2xYT media that include more nutrients used by the bacteria to grow. I found this can sometimes help, especially if you culture the cells for longer time.
For my experiments I changed from IPTG-induction in LB medium to auto-induction (no need for IPTG) with a more "specialized" medium. The E. coli grew better and also the production of protein was enhanced. Below I give you the paper from which I got the auto-induction.
Also, I'm interested, what protein are you trying to express and purify?
Good luck!
Studier, F. W. Stable Expression Clones and Auto-Induction for Protein Production in E. coli. Structural Genomics: General Applications, Methods in Molecular Biology, Chapter 2 1091, 17–32 (2014).
If you have access to E. coli Arctic Express cells you should try it with them. They express chaperons of cryophillic bacteria, which enhances protein expression at low temperatures quite a bit. Additionally replacing the media before induction for a long time period helped me as well.
For me TB medium or auto induction has worked quite well. And as Christophe as mentioned 10-20% is quite good depending on the protein you have.
I once read that increasing the antibiotic concentration before induction, improves plasmid copy number and therefore protein yield. But i have never tried it myself.
While I'm not unhappy with the yields I'm getting, I was expecting higher expression levels, mainly due to the fact that the gene is codon optimized for E. coli expression. Although I am noticing a decent amount of protein consistent with free MBP, which could indicate some in vivo proteolysis and perhaps explain some of the decreased expression?
I'll look into trying the auto induction or TB media as you all have suggested, as well as the E. coli Arctic Express cells. Thank you for your recommendations!
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