Protein expression in foreign cells.

Development of a protein expression system for the target protein is an essential step in the directed he evolution of a protein. Without such a system, it is impossible to separate and characterize the individual mutants produced during the error prone PCR. Cloning of target genes is nowadays a routine technique, however the production of protein in a heterologous host system still relies on a fair degree of luck. This is mainly due to the uncertainties which surround the use or interpretation of the genetic information by host cell machinery that may be different from that in which the gene originated. Foreign proteins may be toxic to host cells, or the protein may be insoluble or suffer proteolytic cleavage. Despite these potential problems, many proteins have been expressed in heterologous host systems, and the constant refinement of protein expression technology is pushing the success rate ever higher.

Many such host cells exist and it is also a good idea to choose a host cell full which the genetic tools are available, which allowed the ready manipulation of the genetic content of the cell. In this way, the process of cloning and expression of the target protein in a foreign, or correctly speaking heterologous, host is greatly simplified. Also, well characterized host cells have been genetically manipulated to reduce common problems associated with the expression of foreign proteins, such as unwanted digestion by proteases.

The host cell of choice for protein expression is the bacterium Escherichia coli, since these cells have several advantages. The organism is extremely well characterized biochemiaclly and is easy to manipulate on the genetic level through the use of DNA vectors specially developed for protein expression. The bacterium grows rapidly in simple culture conditions, and single clones grow in isolated colonies on solid culture media. For these reasons E. coli has successfully been used in many experiments involving directed evolution. Alternatives to the bacterial cell system include yeast cells, and animal cells, however the disadvantage of these eukaryotic systems lies in the fact that the levels of protein expression may very due to factors which are specific to a given cell transformation. This is a serious disadvantage for directed evolution experiments, since it introduces an additional variable during the selection procedure. Indeed, for these reasons, all the experiments using directed evolution in the scientific literature have been based on the use of prokaryotic cells for heterologous protein expression.