What determines a successful protein expression in E. coli?

Question

Some proteins express well in a heterologous host; others- don't. A few requirements are known to determine the protein expression, like a strong promoter (like T7) for transcription and a strong ribosome binding site for translation. I am working with a protein, which consists of 2 subunits - alpha and beta. Both of them are on a plasmid with T7 promoter in front of the beta subunit (i.e. the construct is T7 promoter, CDS for beta subunit, CDS for the alpha subunit). The beta subunit expresses well, but the alpha doesn't. Do you thing this has something to do with the local environment (promoters, RBSs, etc) and how much does it depend on that? How can I increase the protein expression?

Answer 1

I found a very nice paper: Designing Genes for Successful Protein Expression, which covers most factors that determine protein expression. I post parts of it, because I am sure it will be useful to some of you.

Translation can be controlled at the level of initiation and elongation. Initiation of translation is primarily dependent on the sequence of the ribosome binding site (RBS) and early mRNA secondary structure. Other determinants of protein expression are less well understood but equally potent.

1. Initiation of translation

A key component affecting initiation of translation in prokaryotes is the RBS that occurs between 5 and 15 bases upstream of the open reading frame (ORF) AUG start codon. Binding of the ribosome to the Shine–Dalgarno (SD) sequence within the RBS localizes the ribosome to the initiation codon... Affinity of the RBS for the ribosome is a critical factor controlling the efficiency with which new polypeptide chains are initiated. This interaction is in competition with possible base-pairing interactions involving the RBS region that may form within the mRNA itself. Thus, SD sequences with weaker base pairing to the ribosome are more susceptible to interference from mRNA structure. However, some experiments suggest that SD sequences with too strong affinity can be deleterious, particularly at lowering temperatures, by stalling initial elongation. Also critical is the distance between the RBS and the start codon with 5-7 bases from the consensus SD AGGAGG being optimal.

Numerous lines of evidence suggest that the initial 15–25 codons of the ORF deserve special consideration in gene optimization. Studies have shown that the impact of rare codons on translation rate is particularly strong in these first codons, for expression in both Escherichia coli and Saccharomyces cerevisiae. In E. coli, peptidyl-tRNA drop-off during translation of the initial codons appears to be accentuated by the presence of rare NGG codons. These effects appear to be independent of local mRNA secondary structure. It is also true that expression may be recovered by 5' sequence replacement even for sequences that do not show especially strong mRNA structure or contain rare codons or other obvious deleterious elements in this region.

2. Codon bias

The second way in which host codon frequencies can be used is to match the host codon frequencies in the designed gene. This can be done simply by choosing each codon with a probability that matches the host codon frequency...Using sets of genes broadly varied in gene design features, Welch et al. found that variation in synonymous codon usage frequencies had a profound effect on the amount of protein produced in E. coli, independent of local 5’ sequence effects. Variation of at least two orders of magnitude in expression was seen due to substitution beyond the initial 15 codons of the ORF. This variation was strongly correlated with the global codon usage frequencies of the genes, although the codon frequencies found in the highest expressed variants did not correspond to those found in the genome or in highly expressed endogenous genes of E. coli. Multivariate analysis showed that the frequencies of specific codons for about six amino acids could predict the observed differences in expression. It is not clear what the biochemical basis is for this correlation.

3. mRNA structure and translational elongation

While much evidence suggests that mRNA structure can interfere with translational initiation in both prokaryotes and eukaryotes, the effects of structure on elongation are less well understood. This in part may be due to intrinsic helicase activity of ribosomes, which allows translation through even very strong hairpins and may preclude many structures from limiting the translation rate in either prokaryotes or eukaryotes. Perhaps more importantly, mRNA structure is difficult to predict, particularly for actively translated messages which are in continuous flux between various folded and unfolded states.

4. Protein-specific factors providing additional complexity

The protein may be particularly unstable in the host, especially if it is poorly folded due to inherent instability, lack of sufficient prosthetic factors, or improper post-translational modification... Expression of secreted and membrane proteins may be limited by mechanisms for directing these proteins to the membrane. It is even possible that the protein amino acid sequence may limit translational efficiency. For example, proline is thought to be slowly translated in E. coli, regardless of which codon is used.

Expression of the protein may be toxic to the cell leading to instability of the expression vector or host suppression of protein synthesis...A common strategy to reduce toxicity is to lower expression to tolerable levels. Promoters varied in strength can be valuable tools for finding an optimal expression rate for maximal yield...One potential way to avoid toxicity of some proteins is to direct expression to the periplasm or media. This may be accomplished by N-terminal fusion of a secretion signal sequence.

For more information, please read the whole paper. I also recommend reading Design parameters to control synthetic gene expression in Escherichia coli.

Answer 2

Have you tried putting your two genes on two separate plasmids (with different origins of replication and antibiotic selection, of course) and co-expressing that way? If the first of the two subunits is expressing well and the second isn't, that's probably because the ribosomes are falling off the mRNA before fully transcribing the second gene. Are the genes eukaryotic in origin? I think it is difficult to "trick" E. coli ribosomes into treating heterologous sequences as an operon, but maybe someone with more knowledge of prokaryotic translation could help.

Actually, just adding a second T7 promoter in front of gene 2 would probably help quite a bit:

Genes are transcribed either from individual promoters or from a single promoter, leading to a long polycistronic mRNA. Polycistronic expression has been reported to lead to lower expression of the more downstream encoded protein, which can be exploited to influence the stoichiometry of a protein complex (9). Several-fold higher expression with individual promoters compared to polycistronic transcription has been reported (10). (Source)

I do know from personal experience that co-expression using two plasmids can work very well with the right genes!

Answer 3

Mad Scientist covered potential codon-bias issues (which can be ameliorated with Rosettas), but more generally, I've seen tremendous variability in expression rates between different vectors (pET-28 vs pET-24) without any apparent reason. Our lab has had tremendous success with IPTG-inducible vectors (going from pBC-SK to pET-24 increased expression 50-fold).

Beyond expression, the protein can be lost when preparing the cell-free extract. It may not be in the cell pellet if exported courtesy of a signal peptide, or may be discarded in the "debris" pellet when spinning down lysed cells if it's poorly soluble. I've heard a theory that solubility issues may be exaggerated by vectors that saturate the export machinery, which can both impede synthesis and/or be so effective they just cause precipitation. The pET System Manual suggests longer (overnight) expression times at lower (15-20 °C) can help solubility issues. Whatever the reason, engineering away the signal peptide drastically increased expression for many of our proteins.

Answer 4

One important aspect when expressing a protein from a different organism in E. coli is that the codon usage of the original organism is likely different from the codon usage of the E. coli used for expression (Codon usage bias).

Though the genetic code is degenerate, not all codons are equal. They might encode for the same amino acid, but organisms tend to favor specific codons over others and the tRNAs for those codons tend to be present in different concentrations. When you now have a lot of codons in your sequence that are very rare in E. coli, the expression will suffer because the tRNA for those codons is not present in high enough concentrations to sustain the translation.

There are two ways of compensating for that, either you get your E. coli to produce more of the rare tRNAs, or you optimize your sequence to use different codons. For the first solution you can buy certain E. coli strains that contain plasmids encoding the tRNAs that are rare in E. coli, one of those strains is e.g. the Rosetta BL21 strain.

For optimizing the codon usage there are several companies offering to synthesize optimized sequences. There are also tools available online, but I have no direct experience with those.

The optimization is also not as easy as just using the most prevalent codon every time, it has been shown that optimizing the codons to match the translation speed in the original organism can enhance the expression yields. For some proteins, pause sites during the translation might be necessary to ensure proper folding. If the protein doesn't fold correctly it will be quickly degraded and your yield will suffer. This is described in the article "Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host" from Angov et al.

You'll find a nice overview about the whole topic in the article "You're one in a googol: optimizing genes for protein expression" from Welsh et al.