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May 1, 2010 (Vol. 30, No. 9)

New Tools Facilitate Protein Expression

Innovative Solutions Seek to Remove Difficult-to-Express Tag from Recombinant Proteins

  • Polyketide Challenges

    Polyketides are natural products produced by plants, fungi, and bacteria that represent a diverse class of compounds with a broad range of activities useful for many applications ranging from anticancer agents to antibacterials. However, these small molecules are so sufficiently complex that they cannot be easily synthesized through organic chemistry. To produce them recombinantly has also presented significant gene-expression challenges.

    Blaine A. Pfeifer, Ph.D., assistant professor of chemical and biological engineering at Tufts University, is working to overcome these hurdles. “In the last 15 years, there has been a steady effort to produce polyketides in engineering-friendly organisms such as E. coli. The problem is that many of the polyketide synthetases are large proteins (>300 kD) that have unique assembly characteristics.”

    Dr. Pfeifer says pathways to express polyketides are often very complicated. “There can be up to 20 coordinately expressed genes that are required for complete biosynthesis. Some of the larger protein products may be dysfunctional when produced, further complicating the issue. We are finding that the typical rules of molecular biology need to be ‘bent’ to succeed.”

    As an example, Dr. Pfeifer has targeted production of a polyketide that is normally purified from specific strains of soil-dwelling bacteria. “Our case study features introduction of 17 genes into an E. coli expression system. To do this requires a number of optimization steps such as engineering different promoters, optimizing codons, adding a chaperonin, and even adjusting the temperature for E. coli growth. Despite these challenges, we succeeded in producing our compound of interest.”

    Aside from being able to produce polyketides recombinantly, another important advantage of heterologous production is the ability to engineer new derivatives. “By modifying and re-engineering the products, we may be able to produce new compounds, such as modified antibiotics, that have increased potency. A key goal would be to leverage our recombinant production platform to produce new compounds against, for example, antibiotic-resistant bacterial pathogens.”

  • Human In Vitro Translation

    Click Image To Enlarge +
    Thermo Scientific Pierce human in vitro translation technology synthesizes protein in human cell-free extracts. The human translation machinery imparts superior functionality compared to alternative systems such as rabbit, E. coli, or wheat germ systems, the company reports.

    Some researchers opt for cell-free protein expression, i.e., in vitro translational systems. Benefits include compatibility with microliter-scale reactions and faster expression, since traditional cell-based expression can take from days to weeks. However, current in vitro expression systems suffer from low yields or the inability to include post-translational modifications such as glycosylation, according to Brian Webb, Ph.D., platform manager of proteomics R&D at Thermo Fisher Scientific.

    “Typical systems such as wheat germ or E. coli cannot glycosylate proteins. Other mammalian systems such as rabbit reticulocyte lysates in combination with canine microsomal membranes produce low amounts of protein and are not very efficient at glycosylation.” 

    Thermo Scientific has developed an in vitro system derived from immortalized human cell lines that provides biologically active proteins with up to a 15-fold increase in expression. “Initially, researcher’s cDNA is cloned into the kit’s expression vector followed by expression using kit reagents. In 90 minutes, our system can yield about 30 micrograms per milliliter of full-length, functional protein that is relatively clean and can be easily analyzed via Western blot.”

    One application is the simultaneous study of numerous mutant variants in a microplate or expression of large quantities of a single protein for future experiments. Other uses include enabling expression of toxic proteins that cannot be produced in live cells, analyzing protein-nucleic acid interactions, and studying protein complexes.

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