Development and use of engineered peptide deformylases in chemoenzymatic peptide synthesis

PhD ceremony: Ms. C. di Toma, 12.45 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Development and use of engineered peptide deformylases in chemoenzymatic peptide synthesis

Promotor(s): Prof. D.B. Janssen

Faculty: Mathematics and Natural Sciences

The goal of the research reported in this thesis is to explore the potential of the use of peptide deformylase in chemo-enzymatic peptide synthesis. PDF is suitable for the selective N-terminal deformylation of certain N-formyl-peptides without concurrent peptide bond hydrolysis. The challenge of applying PDF industrially prompted us to focus on the development of a new purification method to remove proteins with peptidase activity from the PDF. The use of enzyme engineering in combination with anion-exchange chromatography was proved to be a cost-efficient and industrially applicable purification method. Other aspects such as improving the stability and modifying the substrate specificity were also investigated. In order to broaden the specificity of PDF, we decided to use saturated mutagenesis at positions that play an important role in substrate recognition. A fast assay for the rapid selection of improved peptide deformylase variants in E. coli colonies was developed to analyze the constructed libraries. Various clones displaying new substrate specificity were recovered, sequenced and partially characterized.