there seems no induction of my recombinant protein - (Mar/30/2005 )
hi there! I'm puzzled these days since I've been inducing a recombinant protein construct for certain times, but there seems no obvious induction band.
The gene constructed is a full length one ,and it's coding a transmembrane kinase,about 75kD. What I use is the pET28a system and the BL21(DE3)strain. The construct is no wrong as the sequencing result suggested. And the inserted gene is in frame. I've tried two final concentration of IPTG as 1mM and 0.4 mM, and the induction time from 1h to 20h.But there still seems no obvious band on the gel.
Could any friend tell me whether I was wrong at some place or what should be taken into account that I've missed? Thanks a lot from the bottom of my heart!
Do your protein have rare codon? Change cell strain if it is
thanks a lot, i will have a try.
In my experience, some proteins will not express in BL21(DE3), even if it's the most common strain for bacterial expression. Luckily in our labs, we have different strains of BL21: DE3, pLysS, AI, codon+, etc.... And to my surprise, depending on the culture media (LB, minimal M9) my protein will or will not express in these strains depending on the culture media content, and depending on the strain. So my advice, try different strains. Also, varying the temperature or the induction time, or the IPTG din't have much effet on my protein's expression. It's really a matter of strain. Good luck.
I spent months struggling with my protein. I followed what a postdoc did exactly and it didn't work for me! I expressed another protein successfully, and postdocs watched me to prove my technique was OK. This is a summation of what I learnt trying to express it (it was a very long time)!
Is there any expression at all? Lowering temperature e.g. 30 degrees, 25, or lower can increase expression if it is expressing but not much.
Different strains of E.coli can help, if there are S-S bonds and these are not being formed there are strains such as origami. If the protein is toxic, then it could be killing off the cells, or slowing their growth a lot (hence reducing temp and IPTG conc). BL21 (DE3) plys S are good for very toxic proteins.
What organism is it from? If eukaryotic then maybe E.coli can't express it and you need yeast expression or insect cells (which I know nothing about), or like the other person said different bacteria have different codon usage.
Where are your cells from? If they are from/derived from a glycerol stock they may not work, in fact are they competent-try testing another protein.
Are you using enough antibiotic? I use 100ug/ml ampicillin after someone suggested 50ug/ml wasn't enough. The gene will be lost from the plasmid if there is not enough antibiotic.
You need fresh transformants each time, as the plasmid can be lost in colonies both on the plate and in solution.
Is your growth medium (LB?) made up right and is it at the right pH? I presume everything is sterile and autoclaved.
My problem was solved by using single fresh transformants, growing my starter culture during the day until A260 is about 0.6, then storing in the fridge overnight for a large scale growth in 500ml LB. You can test each colony for expression and use the best one the next day.
Only grow single colonies, if some in a mixture don't have the gene then they compete with the correct colonies and growth isn't as good.
There can be a lot of dead cells in an overnight culture which can cause problems, I use 10ml starter culture for 400/500ml medium, but some people will only use 1% or less of the volume.
I use 400-500ml LB in a 2L flask to allow sufficient aeration. Baffle flasks which make the medium go v frothy can help some proteins.
It's stupid, but if you don't stain your gel for long enough the bands aren't as clear as they could be, this didn't help my cause. Are you spinning down 1ml culture and resuspending in say 50-150ul buffer depending on cell density. I presume you have loaded enough on the gel and the gel and sample preparation is fine.
The protein seems very big, although I am in NMR where my 25.5KDa protein is large for our group. Maybe you could need to express domains or different constructs if appropriate.