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Biological Chemistry

Hijacking Bacteria’s Heat-Shock Response Could Boost Protein Production

Biotechnology: Researchers improved the solubility and activity of recombinant proteins by coexpressing them with a transcription factor that sparks cell repair

by Erika Gebel Berg
August 4, 2014

PROTEIN FOLDERS
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Credit: ACS Chem. Biol.
Researchers added one DNA plasmid with the gene for a mutant transcription factor (σ32, yellow) and another plasmid with a gene for a protein of interest (POI, green) to E. coli. The transcription factor turns on a promoter that triggers the expression of heat-shock genes, which help the bacteria properly fold large amounts of the protein of interest.
Schematic of a new approach to increase the production of soluble recombinant proteins.
Credit: ACS Chem. Biol.
Researchers added one DNA plasmid with the gene for a mutant transcription factor (σ32, yellow) and another plasmid with a gene for a protein of interest (POI, green) to E. coli. The transcription factor turns on a promoter that triggers the expression of heat-shock genes, which help the bacteria properly fold large amounts of the protein of interest.

In academic and industrial labs, Escherichia coli happily churn out recombinant proteins for researchers. But in many cases, the microbes’ cellular machinery balks at this heavy labor and a large amount of the proteins end up misfolded in unusable clumps. To boost capacity, researchers incorporated a mutant transcription factor into bacteria that unleashes the microbes’ natural arsenal for fighting cellular stress (ACS Chem. Biol. 2014, DOI: 10.1021/cb5004477). The approach increased the solubility and activity of aggregation-prone recombinant proteins and may offer a general method for increasing their yield.

When cells get stressed by heat, cold, oxygen deprivation, or other hazards, they deploy proteins to help repair the cell, a process referred to as the heat-shock response. Some of the first responders are chaperones, which encourage other proteins to fold properly. A common biotech trick for increasing the yield of functional recombinant proteins is to coexpress the protein with specific chaperones. However, the approach is limited. “Each chaperone has its own specific substrates,” so researchers have to find the right one to pair with their protein of interest, says Xin Zhang, a research associate of Jeffery W. Kelly at Scripps Research Institute, California. Kelly’s team wanted to recruit the entire cast of heat-shock response proteins, including chaperones, which they reasoned might improve the yield of any protein a researcher might want to express.

To release the totality of the heat-shock response in E. coli without damaging the cells with heat or other stressors, the researchers incorporated the gene for a mutant transcription factor into a DNA plasmid they could add to bacteria. The transcription factor, σ32, is the key regulator of the heat-shock response: It turns on the genes for the proteins in the cell-repair arsenal. The mutation makes the σ32 protein resistant to degradation by the cell.

The team designed the DNA plasmid so that the mutant σ32 would be expressed by bacteria in the presence of the sugar arabinose. After adding arabinose to a culture of E. coli with this plasmid, the researchers observed an increase in numbers of more than 100 heat-shock response proteins, while no change was seen in a chaperone known to work independently of σ32. The E. coli remained healthy as well.

Next, the researchers added a second DNA plasmid to the cells, this one containing a protein of interest, either retroaldolase, endoxylanase, or transthyretin. They added arabinose to the bacterial cultures and then an hour later stimulated the bacteria to produce the protein of interest. Compared with expression without the mutant σ32, bacteria with the transcription factor produced two and three times as much soluble retroaldolase and endoxylanase, respectively. Because solubility does not always translate into functionality, the researchers further tested the expressed proteins and found that σ32 doubled the amount of properly folded retroaldolase and quadrupled endoxylanase activity. The mutant σ32 expression did not improve the solubility of transthyretin. However, it did increase the formation of tetramers of the protein—its functional form—by 39%.

Coexpression of this mutant transcription factor may help researchers in industry who are struggling to make large amounts of functional biomolecules, says Arthur L. Horwich of Yale University. “It gives people another arrow in their quiver.” He says it would be interesting to try the approach in mammalian cells, which Kelly says is in the works.

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