Some proteins are easier to express than others. For some, just clone them into an E. coli vector and let the bacteria do the work. But for others—such as toxic proteins, membrane proteins, glycosylated proteins, and hydrophobic proteins—getting them to express, fold, and function or crystalize can present a challenge.
Scientists at CHI’s “PEGS” conference, held last month in Boston, were keen to talk about some of the difficulties they faced in expressing challenging proteins. Fortunately, they were just as keen to talk about the solutions they came up with.
Typically, when researchers want to express a protein they turn first to E. coli: transfect the bacteria with the appropriate vector, grow it up, plate it out, select colonies, and grow up cultures of the selected clones. Then the fun of purification begins.
Yet for work on an analytical scale, protein can be generated without the need for cell culture (though this, alas, doesn’t alleviate the subsequent need for biochemistry). Several in vitro translation systems based on bacterial, yeast, plant, insect, and mammalian cell extracts exist to turn DNA or RNA into protein.
These all have the added advantage of being able to express proteins that may be toxic to a cell. Each, of course, has its own pros and cons as well—bacterial- and wheat germ-based systems have higher yields, but these and the rabbit reticulocyte systems aren’t capable of glycosylating the resultant proteins, for example, while insect cell lysates introduce insect-specific post-translational modifications.
There are advantages to expressing human proteins using a human system, foremost among them are that the products will be properly folded and properly post-translationally modified, explained Brian Webb, platform manager at Thermo Fisher Scientific. Many academics have published on making a cell-free protein-expression system from human cell lysates, but to date there is only a single commercial product line based on research from the Riken Institute in Japan and licensed by Thermo.
Thermo’s kits make use of a T7 promoter and an internal ribosome entry site, so that it is not necessary for the resultant RNA to be capped before being translated. One set of kits, based on HeLa cell extract, has been optimized for high yield—currently promising up to about 40 ug/mL in about 90 minutes. Data was presented at “PEGS” indicating that yields of several hundred µg of recombinant protein per mL of reaction are possible, and Webb said that such higher-producing kits should be available in the fall. A second set, based on a hybridoma, is optimized for the expression of glycoproteins.
Multiple proteins can be expressed, potentially allowing protein complexes that have more than one subunit in the same reaction, which could “allow those subunits to form and carry out their function,” Webb added.
Later this year Thermo will introduce a series of expression vectors that include C- or N-terminal flag, HA, or myc tags, to complement the currently available HIS tag vector. Fusion vectors encoding GFP are also in the making.