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.