Gene fusion in protein engineering : design of novel peptides and bifunctional enzymes
Abstract: The gene fusion technique is widely used in protein engineering and in this thesis it constitutes the basis of both the construction of artificial bifunctional enzymes and the display of two novel peptides. Artificial bifunctional enzymes have been used as model systems in the studies of native multifunctional enzymes and proximity effects within these complexes. Two various constructs are described. First, the structural genes of lactate dehydrogenase and galactose dehydrogenase were fused in-frame and expressed in Escherichia coli. In vitro observations showed that the chimeric enzyme was able to recycle the coenzyme NAD(H) with a continuos production of lactate without any externally added NADH. Furthermore, proximity effects were pronounced when diffusion hindrance was applied during the recycling reaction. Secondly, the design of the linker region within the bifunctional construct ß-galactosidase/galactose dehydrogenase was examined. The specific activity of the galactose dehydrogenase part of the complex was increased when longer linkers (9 and 13 amino acids) were used within the connecting region. The sequential reaction was carried out more efficiently when enzymes with longer linkers were used as demonstrated both in vivo and in vitro. The co-expression of the genetically engineered bifunctional enzymes and a corresponding native protein subunit was investigated as a method to influence the association pattern of oligomeric proteins and to improve the stability of the artificial bifunctional proteins. Native galactose dehydrogenase was utilised together with both the ß-galactosidase/galactose dehydrogenase and the lactate dehydrogenase/galactose dehydrogenase constructs. Altered associations patterns were observed in both cases. The peptide, Phe-Glu-Ala-His-Ala-Ser, mimicking the catalytic triad in the active site of many serine proteases was expressed as a fusion within the structural gene of the major coat protein of bacteriophage f1. Hydrolytic activity was detectable whit p-nitrophenol activated esters as substrates and it was further possible to elucidate that serine contributed the most to the catalytic activity and the relative position of serine and histidine had a significant influence on the observed activities. A cadmium binding peptide was selected using the phage display technique using a hexapeptide library and cadmium ions immobilised on a metal chelating sepharose. The selected peptide, His-Ser-Gln-Lys-Val-Phe, was cloned as a fusion to the outer cell membrane protein OmpA. Escherichia coli cells harbouring this construct showed a higher degree of survival in growth media supplemented with up to 1.2 mM CdCl2 when compared with cells not expressing this peptide.
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