Solved: Low Yields in Cell-Free Protein Synthesis

Low yield in cell-free protein synthesis is usually fixable… if you know which part of the system is failing.

The most common culprits are easy to address: template purity, transcription or translation inhibitors, and potassium and magnesium concentration. Check those first. But when the basics are in order and yield is still poor, the problem is usually one of three things specific to the in vitro system itself.

This article covers those three causesL codon bias, poor protein folding, and phage polymerase uncoupling, with specific fixes for each. The examples use prokaryotic E. coli-based extracts, but the folding section applies equally to eukaryotic systems including rabbit reticulocyte and wheat germ extracts.

Problem 1: Codon Bias

Codon Bias is a problem in both in vitro and in vivo expression systems. The problem arises when the protein of interest contains codons for which the expression extract contains few tRNAs (more info here).

For example, the amino acid arginine has four codons, CGU CGC, CGG, and CGA. In E.coli CGA is rarely used (see this E.coli codon usage table and (1)), which means that an E.coli extract will have few tRNAs for CGA. So if your gene contains several CGA codons the ribosome will struggle to “find” enough of the CGA tRNA to continue the synthesis, which can result in low (or even no) yield.

Fix

There are three possible approaches to this problem:

i. Lower the temperature of the reaction to 30C (or even lower). This will slow translation down, allowing time for the correct tRNA to be recruited.

ii. Optimize the codon usage your protein of interest, for example by gene synthesis, to eliminate any rare codons and replace them with more common ones.

iii. Use an expression system that is more suitable (i.e. has a compatible tRNA composition) for your gene. A eukaryotic-based system is one option.

Problem 2: Poor folding

It is common to observe higher protein yields with bacterial systems than with eukaryote systems. But sometimes more protein does not mean functional protein because even after successful protein expression, the job is not done; your protein still needs to fold properly to be functional.

And folding problems can occur due to incompatibilities between the host system and the protein bring expressed. For example, some proteins require post-translational modification, chaperones, or even disulfide bridges to fold properly, which cannot be provided by E.coli.

Unfolded protein form intermediates that aggregate. If the protein requires post-translation modifications, aggregation is very likely.

Fixes

i. Try adding protein folding factors (chaperones) to the in vitro extract may help (2).

ii. For proteins that require post-translational modifications, use rabbit reticulocyte system (with canine microsomal membranes) (RRL) or wheat germ extract (WGE) would be a better choice than E.coli. Differential folding is sometimes observed between RRL and WGE for the same protein. RRL tends to produce active protein than WGE in most cases (3).

iii. It is also sometimes possible to re-fold proteins after they have aggregated. Click here for more info.

Problem 3: Phage Polymerases

The in vitro coupled transcription/translation systems use phage polymerase for transcription. Phage polymerases (T3, T7, and SP6) transcribe at a rate much faster than native E.coli polymerase. The transcription and translation process in E.Coli is coupled, which means that the ribosome closely follows the emerging mRNA strand.

But with phage polymerases, transcription happens much faster than translation by native E.coli machinery. This results in an uncoupling of transcription and translation so the mRNA transcript is exposed, rather than being bound by ribosomes, and is susceptible to degradation or formation of secondary structures. The net result is the overall reduction in protein yield.

Fix

Reduce the speed of phage polymerase transcription. Phage polymerases function optimally at 37C. However, by reducing the temperature of the reaction to 20C, the speed with which polymerase transcribes can be slowed down. This to some extent also improves the coupling between transcription and translation. Lowering temperature has been shown to produce active protein in vitro (4).

These three causes — codon bias, phage polymerase uncoupling, and folding incompatibility — account for a significant proportion of low-yield problems in cell-free expression that persist after the basics have been checked. They’re not always the first thing you’d suspect, which is why yield problems in otherwise well-set-up reactions often go unresolved longer than they should.

If you’re working with proteins that require post-translational modification or disulfide bridges, the folding section is the most relevant starting point. For straightforward bacterial proteins with unusual codon composition, start with codon bias. For reactions where yield drops off over time rather than being consistently low from the start, phage polymerase uncoupling is worth investigating first.

Related articles on Bitesize Bio

Solved: Heterologous Gene Expression Problems —The in vivo version of this article..!

References

1. Kurland, C et al (1996) Current Opinion in Biotechnology. 7, 489-493.

2. Nishimura, N et al (1995) Journal of Fermentation and Bioengineering, 79 (2), 131-135.

3. In vitro protein expression (cell-free expression) Guide (Promega).

4. Iskakova, M. et al . (2006) Nucleic Acid Research. 34 (19), p135.

Shoba

Shoba has a PhD in Biology from the University of North Carolina at Charlotte.

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