Modulation of protein properties and functions by site-specific protein modification.

摘要

Site-specific protein modification has been widely used to incorporate a variety of functional groups into proteins and peptides, and consequently modulate and study their physical properties and biological functions. To achieve site-specific protein modification, many chemoselective ligation reactions have been developed. Among these ligations, native chemical ligation (NCL) and copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) are the most widely used two ligations. In this thesis, we have utilized NCL and CuAAC to functionalize the N-terminal of expressed proteins and peptides with synthetic molecules to alter their physical properties, improve their biological activities and even create some new functions.;In Chapter 2, we present a general approach to increase the solubility of hydrophobic proteins and peptides by site-specific incorporation of a small betaine moiety onto N-terminus via NCL.;In Chapter 3, we present an approach to functionalize proteins' N-terminus with a polymerizable alkene group by NCL. The alkene functionalized proteins can be copolymerized with acrylamide to form protein-polyacrylamide hydrogel.;In Chapter 4, we present two general approaches to form N-terminally conjugated expressed protein dimers and trimers that were difficult to prepare before. The first approach utilizes NCL to conjugate the N-terminus of two monomers with a dithioester linker to form a homodimer. In second approach, we develop a combination of NCL with CuAAC to form protein homo- and heterodimers, and homotrimers.;In Chapter 5, we apply our protein dimer formation approaches, which are presented in Chapter 4, to prepare dimers of HIV fusion inhibitor peptides with enhanced antiviral activities.;Lastly, in Chapter 6, we present the first example of production of homogenously glycosylated glycoproteins with an N-terminal cysteine. As an example, the N-terminal cysteine containing antibody Fc is produced and further labeled with cyclic-RGD peptide for cancer cell targeting. This work has great potential for the development of cancer cell targeting therapy.