Louise Albertsen

September 2002 – April 2009: M.Sc. (Pharm), Faculty of Pharmaceutical Sciences, University of Copenhagen

January 2007 – September 2007: M.Sc. Student, Laboratory of Synthetic Protein Chemistry, Rockefeller University

June 2009 – January 2010: Chemist, Department of Protein and Peptide Chemistry 2, Novo Nordisk A/S

February 2010: Industrial Ph.D student, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen and Department of Protein and Peptide Chemistry 2, Novo Nordisk A/S

Optimization of recombinant expression of peptide thioesters and their application in C-terminal peptide modifications

Background
Naturally derived peptide and protein therapeutics generally suffer from low in vivo stability and inadequate delivery. Unnatural building blocks (e.g. N-methyl amino, D-amino, and β-amino acids) are extensively used to engineer peptide therapeutics in which subtle changes improve their pharmacokinetic and pharmacodynamic properties. Additionally, many endogenous peptide hormones are amidated in the C-terminal and thus can not be directly produced by conventional recombinant techniques. However, full synthesis of such therapeutics is associated with high costs and environmental issues.

Expressed protein ligation (EPL) is a semi-synthetic approach for generation of peptides and proteins that combines the advantages of recombinant and chemical approaches. In EPL, a recombinantly derived peptide thioester is ligated to a complementary synthetic peptide fragment containing an N-terminal cysteine resulting in the formation of a native peptide bond (1,2). The ligation reaction is generally referred to as native chemical ligation (3). The simplicity of EPL has placed the technology in the forefront of protein synthesis with the potential of generating large amounts of protein or peptide. However, a primary disadvantages is the need for a cysteine at the ligation junction. Recently, ligation of smaller peptide fragments by direct aminolysis of the peptide thioester with the α-amino group of another peptide has been achieved (4). Recombinantly derived thioesters has further been used to make amidated peptides by treatment with ammonia (5).

Project
The present project will focus on the development of an intein-based expression and purification system for the large-scale production of peptides with a reactive C-terminal thioester group prone for further chemical processing. Using endogenous peptides as models, expression and purification of the corresponding thioester derivatives will be optimized by varying relevant factors like construct design, culture and expression conditions, and intein selection and removal. The thioester derivatives will subsequently be converted to either C-terminal amides or C-terminal extended analogs by amidation or chemical ligation, respectively. The ligation strategy will be based on native chemical ligation or direct aminolysis, which both create a native peptide bond between the merged fragments. The chemistries used for amidation and ligation will be explored and optimized in model studies using small synthetic peptide fragments prior to applying it on the recombinantly derived peptide thioesters.

References

1. Muir T.W., Sondhi D., and Cole P.A. (1998) Proc. Natl. Acad. Sci. USA, 95, 6705-6710

2. Muir T.W. (2003) Annu. Rev. Biochem. 72, 249-289

3. Dawson P.E. et al. (1994) Science. 266, 776-779

4. Payne R.J. et al. (2008) Angew. Chem. Int. Ed. 47, 4411-4415

5. Cottingham I.R. et al. (2001) Nat. Biotechnol. 19, 974-977

Main Supervisors:
Kristian Strømgaard, PhD.
Professor
Department of Medicinal Chemistry

Jens Chr. Norrild, PhD.
Principal Scientist
Novo Nordisk A/S, Department of Protein and Peptide Chemistry 2

Co-supervisor:
Allan C. Shaw, PhD.
Senior Project Manager
Novo Nordisk A/S, Department of Protein Expression