TY - BOOK

T1 - Processing Proteases

AU - Ødum, Anders Sebastian Rosenkrans

PY - 2015

Y1 - 2015

N2 - Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N-terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease suitable to leave native C-termini.During this study the substrate specificity of LysN was profiled with a synthetic fluorogenic peptide library which allowed for kinetic characterization. A novel profiling method using encoded beads was proposed to efficiently profile human Renin. Recombinant expression was explored with Pichia pastoris as the preferred expression host to produce functional rAm-LysN. Site-directed mutagenesis and randomized mutational approaches were used to introduce mutations in order to alter the wildtype specificity. Finally, with the knowledge of preferred substrates we showed that recombinant LysN could remove functional tags yielding native C-termini of twelve model peptides. The results of this study take an important step towards exploiting the unique features of LysN for industrial processes and will allow for novel ways of using fusion technologies in the production of recombinant proteins.

AB - Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N-terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease suitable to leave native C-termini.During this study the substrate specificity of LysN was profiled with a synthetic fluorogenic peptide library which allowed for kinetic characterization. A novel profiling method using encoded beads was proposed to efficiently profile human Renin. Recombinant expression was explored with Pichia pastoris as the preferred expression host to produce functional rAm-LysN. Site-directed mutagenesis and randomized mutational approaches were used to introduce mutations in order to alter the wildtype specificity. Finally, with the knowledge of preferred substrates we showed that recombinant LysN could remove functional tags yielding native C-termini of twelve model peptides. The results of this study take an important step towards exploiting the unique features of LysN for industrial processes and will allow for novel ways of using fusion technologies in the production of recombinant proteins.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122770738105763

M3 - Ph.D. thesis

BT - Processing Proteases

PB - Department of Chemistry, Faculty of Science, University of Copenhagen

ER -