SFI UREKA Site in Molecular Recognition
Translating Design into Application

Biophysical Characterisation

Project 10:      Expression, Purification and Affinity Characterisation of Carbohydrate-Binding Single-Chain Variable Fragment Antibodies

Supervisor:     Prof. Robert J. Woods, School of Chemistry

Project Summary:  The Woods group employs computational and experimental techniques in the study of carbohydrate-protein interactions. In this project, experimental techniques will be employed to study interactions between carbohydrates (CHOs) and single-chain variable fragments (scFvs).  CHOs on the surface of many infectious pathogens and tumor cells are desirable targets for therapeutic agents, such as scFv, because they are often abundant, unique structures and are the main virulence factor for several pathogens.  scFvs are among one of the most popular types of recombinant antibody (rAbs) fragments as they have been successfully modified into a number of different antibody formats and are easily expressed by several expression systems, e.g. in E. coli.   scFvs are small rAbs that contain the complete antigen binding site, which includes the variable heavy (V _H) and variable light (V _L) domains, of an antibody that are linked together by a flexible polypeptide linker (e.g. (GGGGS) ₃). 

The Woods group is currently working to characterize scFvs that bind to different antigenic bacterial and yeast antigenic CHO targets, such as the Group B Streptococcus type III polysaccharide ¹, and  b-1-2-mannotriose on Candida albicans ², respectively.  Determining the specificity and affinity of scFv for their CHO targets is crucial to determining their effectiveness as potential therapeutic agents.  In this research project the student will use E. coli to express different anti-CHO binding scFv.  The student will then purify the scFv by immobilized affinity chromatography and perform initial specificity analysis to structurally related CHO antigens by ELISA and western immunoblotting.  Detailed affinity measurements and cross-reactivity (specificity) analysis will finally be conducted by Biacore analysis.

The student will work closely with Dr. Nina Weisser on a daily basis and will be trained in a range of laboratory skills.  The student will learn detailed aspects of protein analysis research, including protein expression, purification and affinity analysis and will gain an appreciation for different types and ranges of CHO-protein interactions.  The student will receive training that will be beneficial in many different fields of science and will gain that knowledge and confidence to executed detailed scientific analysis.

References [1] Weisser NE, Almquist, KC, Hall. JC. 2007 Vaccine. 25, 4611-4622. [ 2] Han Y, Riesselman MH, Cutler JE. Infect Immun. 2000 68, 1649-1654.

• Disclaimer
• Privacy
• Copyright
• Accessibility