In my last article, I introduced Cell-Free Protein Synthesis.

Today I want to talk about a major bottleneck in in vitro cell-free protein expression; low yield.

Most often, paying attention to the important factors such as template purity and design, transcription and translation inhibitors, and potassium and magnesium concentration will solve any problems with low yields. But, sometimes it’s more complicated and other approaches are needed.

Here are some problems, along with possible solutions that could solve your problems of low yield when using prokaryotic in vitro extract systems.

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.


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.


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.


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).

Although this is not an exhaustive list of reasons for low protein yield in in vitro synthesis, these tips should solve a fair number of problems.

Do you know of other reasons for poor yield? Please drop us a line in our comments section to share it.

Related articles on Bitesize Bio

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


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.

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