One of My Undergraduate Projects

“A large number of therapeutically useful cyclic and linear peptides of bacterial or fungal origin are synthesized via a template-directed, nucleic acid-independend nonribosomal mechanism. This assembly line mechanism is carried out on megaenzymes called nonribosomal peptide synthetases (NRPSs). They are organised in iterative modules, one for each amino acid to be built into the peptide product. Usually the order of such modules in NRPSs is colinear to the sequence of the synthesized peptide, exploring an assembly line way comparable to a linear workflow. A typical module usually comprises about 1000 residues and is responsible for one reaction cycle of selective substrate recognition and activation as adenylate, covalent intermediate fixation as enzyme-bound thioester, and peptide bond formation.” (Text and graphic taken from Marahiel Website)

Tyrocidine synthetase TycA is one of many enzymes involved in the non-ribosomal synthesis of the peptide antibiotic tyrocidine. The research groups of both Marahiel (Marburg, Germany) and Walsh (Harvard) have been at the forefront of non-ribosomal peptide synthesis for many years, and have beautifully elucidated how individual building blocks are assembled in a thio-template mechanism (see sidebar).

The tycA DNA sequence is highly conserved and is part of a larger operon coding for various other components of the enzyme complex. The goal was to provide for larger amounts of tyrocidine synthetase A.

Therefore, I amplified the DNA fragment encoding the tycA by PCR and cloned it into the plasmid pQE60. This plasmid was transformed into XL1blue cells and overexpressed. The resulting protein was purified by immobilized metal chelate affinity chromatography, which binds the Tyc A protein through its terminal histidine tags.

This project has given me hands-on experience in techniques commonly applied in microbiology and related fields.