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June 5, 2018

Genetic Engineering and Directed Evolution Improve Stability of Therapeutic Proteins

Source: Thomas Shafee/Wikipedia, CC BY 4.0

  • Scientists at the University of Texas at Austin say they have developed a new technique to make therapeutic proteins more stable. They believe their advance will improve the drugs' effectiveness and convenience, leading to smaller and less frequent doses of medicine, lower healthcare costs, and fewer side effects for patients with cancer and other diseases.

    Their study (“Custom Selenoprotein Production Enabled by Laboratory Evolution of Recoded Bacterial Strains”) appears in Nature Biotechnology.

    “Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered β-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely,” write the investigators.

    “We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.”

    Many drugs commonly used to treat cancer and diseases of the immune system, including insulin, human growth hormone, interferon, and monoclonal antibodies, can have a short active life span in the human body. That's because these drugs, which are proteins or chains of amino acids linked together by chemical bonds, contain the amino acid cysteine, which makes chemical bonds that break down in the presence of certain compounds found in human cells and blood.

    The new method replaces cysteine with the amino acid selenocysteine, which forms hardier chemical bonds. The change would lead to drugs that have the same therapeutic benefit but increased stability and may survive longer in the body, according to the new study.

    "We have been able to expand the genetic code to make new, biomedically relevant proteins," said Andrew Ellington, Ph.D., associate director of the Center for Systems and Synthetic Biology and a professor of molecular biosciences, who co-authored the study.

    Biochemists have long used genetically modified bacteria as factories to produce therapeutic proteins. However, bacteria have built-in limitations that previously prevented harnessing selenocysteine in these therapies. Through a combination of genetic engineering and directed evolution, whereby bacteria that produce a novel protein containing selenocysteine can grow better than those that don't, the researchers were able to reprogram a bacteria's basic biology.

    "We have adapted the bacteria's natural process for inserting selenocysteine to remove all the limitations, allowing us to recode any position in any protein as a selenocysteine," said Ross Thyer, Ph.D., a postdoctoral researcher in Ellington's lab who led the study.

    The team described the basic method in a paper in the Journal of the American Chemical Society in 2015. In this latest study, the team demonstrated the practical application of this method by producing medically relevant proteins, including the functional region of the breast cancer drug Herceptin® (trastuzumab). The team showed that the new proteins survive longer in conditions similar to those found in the human body compared with existing proteins containing cysteine.

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