On bacterial formats in protein library technology

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for all known forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes in food industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, a number of techniques, all collectively referred to as ‘protein engineering’, are performed in the laboratory. Traditionally, methods involving ‘rational design’, where a few alterations are introduced at specific protein locations to hopefully result in expected improvements have been applied. However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, from which rare clones showing enhanced properties can be isolated have contributed greatly to the field of protein engineering. In the present thesis, different protein traits of biotechnological importance have been addressed for improvements by the use of such methods, in which there is a crucial need to maintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterial cell-membrane compartmental confinement. In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genes encoding variants showing an enhanced soluble expression of the enzyme frequently used in biotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein (GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles, was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay (PCA), based on the affinity driven structural complementation of a genetically split βlactamase enzyme was used to identify variants having desired target protein binding abilities, including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to the human cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.