ABSTRACT
The most appropriate methods of expression and purification are determined by the experiments that one intends to perform on the protein of interest. For experiments requiring large quantities of the protein, for example 1antitrypsin replacement therapy, then bacterial1 or yeast expression may be most appropriate.2 If the material is to be given to humans then stringent quality assurance is required and the absence of human-infecting viruses in E. coli and yeast makes them attractive. If the protein is to be used in an animal model, then expression in yeast has the advantage that yeasts do not produce endotoxin. E. coliderived endotoxin contamination of test proteins can lead to problems interpreting data in animal and cell culture experiments.3 The convenience and high yields from E. coli mean that protein crystallographers often try bacterial expression first; however, if the protein of interest has a complex structure, is prone to aggregation, needs proenzyme processing, or requires highly specific glycosylation
in a higher organism.4,5 Yeast may be sufficient; however, insect or mammalian cell culture are likely to give better protein products.6 Whole organism expression, such as secretion of recombinant protein into transgenic ovine milk, has been used where the combination of large quantities of product must be combined with the need for eukaryotic processing of the recombinant protein.7,8
At the opposite end of the scale from whole organism expression are the new commercial kits that allow protein expression in a cell-free system.9 These kits conveniently produce small quantities of protein, sufficient to test for binding affinity or enzymic/inhibitor activity but the high cost prohibits scaling up even to the milligram range. Template DNA is usually plasmid-derived, however the generation of template DNA using PCR allows for flexible protein expression using sequence derived from genome databases and cDNA libraries.
