Platforms for in vitro glycoengineering of proteins

Protein glycosylation is an important post-translational modification that affects the protein structure and function. Glycoengineering is the process where the glycosylation outcome of the protein is modified for a designated purpose such as functionalisation of the glycans or generating a glycan-defined glycoprotein. The biggest advantage of in vitro glycoengineering methods is the controllable physical parameters that define the reaction environment.In this dissertation, three different aspects of in vitro glycoengineering approaches were addressed; 1) the design of a DNA-guided protein scaffold for multi-glycosyltransferase reactions (Chapter 2), 2) immobilisation of glycosyltransferases on a stationary column for re-usable, on-column glycosylation (Chapter 3), and 3) application of in vitro glycoengineering principles to a cancer -specific IgM antibody which was firstly characterised for its site-specific glycosylation (Chapter 4), the knowledge of which was used for multi-purpose conjugation of specific IgM glycans (Chapter 5).In Chapter 2, the design of a sequence specific DNA-binding protein with a monomeric streptavidin fusion protein adaptor (TALE-mSA) was presented. By tailoring the DNA binding sequence of the adaptor to a predefined custom DNA program, spatial alignment of streptavidin-tagged glycosyltransferases can be achieved. TALE-mSA adaptor proteins were recombinantly expressed and purified from E. coli and tested for DNA binding capacity.In Chapter 3, recombinantly expressed human B4GALT1 containing an N-terminal His-tag was used as a model glycosyltransferase to explore the possibility of in vitro glycosylation by immobilised of a glycosyltransferase on Ni-NTA resin. Galactosylation of de-sialylated, de-galactosylated bovine fetuin was found to be rapid; approximately 70% of the glycans were optimally galactosylated within 30mins. Re-usability, and adaptability for sequential on-column glycosylation were explored. IVIn Chapter 4, in-depth site specific glycosylation characterisation was performed on an anti-cancer IgM antibody, PAT-SM6, to establish the baseline glycosylation for the in vitro glycoengineering described in Chapter 5. As approximately 50% of the glycans contained a free galactose, a single step sialylation by ST6GAL1 using an azide-labelled CMP-sialic acid was chosen for chemo-enzymatic functionalisation of IgM glycans by click chemistry. In-depth characterisation of the incorporation of the azide-functionalised groups was determined.In conclusion, development and application of these platforms will provide more tools towards understanding the details of how glycosylation affects protein function.