The three most important things when expressing protein for high-throughput applications are: optimize, optimize, optimize. Put the best genes you can into your expression system, use the most fitting expression systems, and make sure the growth conditions are suited to achieve the most from those systems.
Whether it’s drug screening, crystallography, NMR, mass spectrometry, or binding and toxicity studies, the earliest decisions about choice of template and how to express it can have ripple effects throughout a project, points out Frank Schäfer, Ph.D., associate director of R&D, head of DNA and protein sciences at Qiagen.
One problem researchers frequently encounter when trying to express human proteins in bacterial cells is, for example, that the organisms use the genetic code differently. True, the genetic code is universal in that the same three-nucleotide codon always signifies the same amino acid. But there’s ambiguity in the other direction—that is, most of the 20 naturally occurring amino acids can be encrypted by more than 1 of 61 possible codons, and different organisms prefer different spellings of those amino acids.
There are even some sequences used by one organism that may be misinterpreted by another—take, for instance, the presence of mammalian intragenic sequences that mimic bacterial ribosomal entry sites.
Organisms display intraspecific codon usage biases as well. Highly expressed proteins are often spelled with a skewed set of otherwise synonymous codons relative to more lowly expressed proteins. Certain tRNAs are more abundant than others, while some tRNAs may be recharged more rapidly than others—and the two sets are not always the same.
Thus there is even plenty of room for improvement expressing a native protein in its endogenous environment, let alone in an exogenous system. “It seems that the genes as they have been made by evolution are not optimized for maximum expression,” says Dr. Schäfer, who, along with the others included in this article, will be speaking at CHI’s “PepTalk” to be held in San Diego later this month.
“By optimizing the codon usage and several other parameters, we were very successful in dramatically increasing expression of human genes in human cells,” Dr. Schäfer explains.
An algorithm designed by Geneart (www.geneart.com) allows Qiagen to offer a complete set of plasmids (termed QIAgenes) covering the entire annotated human genome, optimized for expression in either E. coli or human/insect cells. QIAgenes are designed to take into account the host cell’s preferred codon usage as well as mRNA secondary structure and stability considerations, and contain a 6xHIS tag to facilitate purification. QIAgenes plasmids can be used as templates for cell-free protein expression as well.
Rational gene design can increase the chances of successful expression and can yield as much as a 50-fold increase in protein expression. This, in turn, can make a huge contribution to the whole project, notes Dr. Schäfer. “The more protein you have, the easier it is to perform all of the downstream experiments.”