I have a insect protein which contains 4-6 disulfide bonds in its active form. I want to use a bacterial expression system (or yeast if it does not work out for bacteria) for over-expression of a properly folded protein in good yield. We know that e. coli systems are not suitable for such proteins and there are several methods to use to make active protein even in e. coli. It includes protein tag with signal peptide for periplasm/secretion or specific strains which do have less reduced environment (certain gene-silencing) to promote disulfide formation. My question to the community is what worked for you if you have ever faced with this task? Any specific strain, expression system, specific vectors, expression conditions, buffers or any peptide signaling tag anything. I'd be obliged to for your valuable response and suggestions.
If you protein natively carries a signal sequence then you might have a good chance that you can secrete it to the periplasm of E. coli and get properly folded protein. You might however need to replace the signal sequence with a bacterial one. If it does not natively have a signal, then the probably is low that you can secrete it in E.coli. You could also try the SHUFFL strains that NEB sells which permit disulfide bond formation in the cytoplasm. There is no predicting which will work better, you just need to try. Have you considered just using a baculovirus insect expression system?
Dear Chetan
as michael told you, expecially if the mw of your protein is not very high secretion on the periplasm by and a pelb or ompa secretion sequence at the n terminal can be a possibility. it worked very well for me ti produce human Cu,Zn sod1. alternativelly you cam try to perform citoplamic expression in strain as the shuffle or the origami which are missing some citoplamic protein involved in disulfide reduction as trx and grx and favour the formation of s-s.
sincerelly 4-6 s-s seems quite an hard work for e.coli and probably in your piace i will evaluate also an ekautiotic expression system. Actually i'm usg the expi293,
https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expi293-expression-system.html
which is quite expensive but very powerfull in term of post translational modification, expression yields and complexity of the protocool the is very simple and reproducible.
From my point of view when the price of this system will decrease thia system will make useless the use of other eucariotic expression systems as pichia or baculovirus.
good luck
Manuele
You might find the following referenced article useful.
Evelina Angov, Collette J Hillier, Randall L Kincaid, Jeffrey Lyon, January 2008. Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host. PLoS ONE 3(5):e2189.
The proteins in question were from a eukaryotic organism and were about 45 KDa, each containing 3 disulfide bridges. The expression host was E. coli. All proteins were expressed in soluble form and appeared to be structurally correct.
The approach we used had two parts, but it may be that you only need to use one of them.
Before I describe those, allow me to talk a bit about translation.
Based on the work in this paper, in subsequent papers, and in the work leading to this paper, it is my opinion that protein translation is not a steady state process, but it varies regionally according to the structure of the subdomain currently in translation. Thus, translation pauses from time to time as the translation of one subdomain ends and the next begins. If this is true, this translational pausing may allow the nascent subdomain to fold prior to beginning translation of the following domain and helps to deter the formation of misfolded protein aggregates.
It therefor follows that for heterologous protein expression, it may be important to slow things down.
The easiest thing for you to try is expression at a controlled, reduced temperature, and to control the beginning of expression by using a tightly regulated promoter/repressor system. We used the lac repressor and induced with IPTG. But there are two important steps that are often overlooked. 1. Prior to induction we grew the biomass (at 37C) in the presence of 0.5% glucose. This insured that that promoter was minimally active while cells were growing. 2. Before adding the IPTG we reduced the temperature of the culture to our target induction temperature of 30 C. This insured that the beginning of synthesis occurred at 30C rather than 37C and is crucial.
The other element of our approach was to prepare synthetic genes. It required that we know the frequency of appearance of every codon in our heterologous organism and in E. coli. Our synthetic genes were designed such that codons appearing frequently in our heterologous gene were represented by frequently appearing codons in E. coli. The same for moderate frequency and low frequency codons. If you don’t know the codon usage patterns for your heterologous organism, you can’t use this method.
Our experience was that not all organisms require “codon harmonization” to achieve high level soluble expression. Paying close attention to the expression conditions was enough.
Dear Jeffrey,
Thank you for your reply. It looks like a good point to start as you mentioned and based on references regarding your post. If I get some positive result, would it be okay to consult with you for better results/plan? Again thanks for your helpful insight.
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