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Oct 1, 2009 (Vol. 29, No. 17)

Protein Expression Poised for Reinvention

Fast-Breaking Developments Drive Need for Constant Introduction of Novel Technologies

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    The PERLXpress system from Chesapeake PERL combines well-established recombinant baculovirus technology with mass oral infection of synchronous Trichoplusia ni larvae.

    Novel fluorescent tags, new insight into protein crystallization, and alternative expression systems were among the presentations that grabbed participants’ attention at CHI’s “Bioprocessing Summit” held recently in Cambridge, MA.

    Green fluorescent protein (GFP) reigns supreme as a molecular detection method.  A protein isolated from marine organisms, GFP glows green under wavelengths in the ultraviolet range. When engineered into the sequence of target proteins, it can be used to track cellular events. Geoffrey Waldo, Ph.D., of the bioscience division at Los Alamos National Laboratory, has guided useful modifications of this important technology, working through some of the more vexing issues.

    According to Dr. Waldo, “GFP technology is limited by the fact that existing fluorescent protein tags can perturb protein solubility or may not work in living cells.” One way to get around this is to “split” the bulky fluorescent proteins into smaller pieces that might be less bothersome as tags, but existing split GFPs are poorly folded and interfere with protein behavior. This makes them risky to use for monitoring protein interactions and tagging proteins. To overcome these drawbacks, Dr. Waldo and his colleagues have engineered soluble, cell-associating GFP fragments that perform as exemplary tags.

    “We evolved superfolder GFP 1-10 by DNA shuffling to improve its solubility and increase its complementation with sulfite reductase GFP 11,” Dr. Waldo stated. The group’s strongest candidate protein had an 80-fold improvement over the starting material. The system is simple to use and works in living cells or in the test tube. Further, the tags do not change the behavior of their target proteins.

    Dr. Waldo and his team have used this technology to establish an in situ assay for quantitatively monitoring the aggregation of Tau, the protein associated with neurodegenerative changes in Alzheimer’s patients. This provides a tool for understanding the molecular basis of a number of human neurological diseases.

    The Los Alamos team’s split GFP system reportedly lets researchers screen through millions of cells in a few hours to find useful proteins for targets for drug design. A notable achievement from the researchers is a series of split fluorescent proteins, tiny pieces of which are ideal for screening protocols. “The problem with the conventional split GFP protein is that it is plastered with lots of intellectual property protection,” said Dr. Waldo. “We have isolated and engineered an alternative fluorescent protein panel from a sessile coral that sports purple spines.” 

    With this newly developed scaffolding, an additional tagging option will soon be available to investigators that is free of the intellectual property thicket. “This makes our new split fluorescent protein much more affordable,” stated Dr. Waldo.

  • Protein Crystallization

    The ocean depths offer unique opportunities for uncovering proteins that have evolved under exotic conditions, according to Joseph Ng, Ph.D., associate professor, University of Alabama, Huntsville. Dr. Ng and his coworkers confront the challenges of developing three-dimensional descriptions of protein molecules through the use of x-ray crystallography.

    Current technology takes advantage of structural genomics, in which a sequenced genome is screened for coding regions that can be analyzed through a bioinformatics program. Candidate sequences of interest are then optimized with the goal of expressing proteins and building crystals that can be subjected to analysis.

    In a natural state it would be selectively disadvantageous for an organism’s protein to crystallize spontaneously. For this reason, most proteins are resistant to crystallization under normal physiological conditions. Yet, knowledge of a molecule’s 3-D structure is essential for a rigorous understanding of its structure and function, and for the design of drugs that would influence its behavior in a therapeutic situation.

    Dr. Ng’s program involves identification, cloning, expression, purification, crystallization, and crystallography of promising molecules. “Success depends on the solubility and stability of the protein and determining the optimal nucleation and crystal growth parameters. Problems may arise if the protein solution is heterogeneous, if the molecules lack a compact arrangement and move extensively in solution, or if the molecules undergo nonspecific aggregation. We have a number of tricks in our bag for overcoming these roadblocks.”

    The group has access to a number of hyperthermophilic bacterial proteins, obtained from deep-sea explorations, and these are yielding useful information on the adaptations of proteins to harsh environments. The Huntsville team has found that many of these proteins have a high propensity to crystallize. 

    Not all heat-resilient proteins are good candidates for crystallization, but for those that are, their structures reveal common molecular features. They possess unique architectural and molecular “hot spots”  that endow them with stability and the ability to form crystal lattices. Using high-throughput gene-synthesis and protein-engineering methods developed in Dr. Ng’s lab, proteins that do not crystallize are coupled to hyperthermophilic protein fragments as crystallization vehicles. 

    Protein crystals suitable for x-ray crystallography can now be obtained by this method where they cannot be acquired otherwise. The subsequent processing of the molecules exploits capillary tubes into which the protein solutions are introduced. The tubes are sealed with nail polish, and counter diffusion takes place within the capillary. The crystals are then subjected to analysis in situ. Many proteins have been analyzed including lysozyme, Factor XIII, Con A, insulin, and glucose isomerase.

    “Over 50 percent of targeted proteins in structural genomics programs cannot be crystallized using conventional methods,” Dr. Ng pointed out. “So this approach opens the door to a whole realm that was previously inaccessible.”

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