2017 Winner: Structural Studies to Understand Evasion of an Astrovirus serotype-2 subtype to a Potent Neutralizing Antibody

Project Information
Structural Studies to Understand Evasion of an Astrovirus serotype-2 subtype to a Potent Neutralizing Antibody
Engineering
BME 195
ABSTRACT
A new human astrovirus serotype HastV-2-Oxford, part of a largely variable HAstV-2 family, was found to be resistant to a potent neutralizing antibody PL-2.The protein Spike-2-Oxford was recombinantly expressed in E.coli and purified using Talon batch purification followed by size exclusion chromatography. X-ray crystallography allowed visualizing the three-dimensional structural difference compared to its wild-type countertype Spike-2-CDC-Spain. Programs such as Coot and Phenix were utilized to reach a well-determined structure. The structure revealed that a mutation from Serine463 to Proline463 part of a crucial Spike/antibody contact region was responsible for the antibody neutralization resistance in loop 1. Resistance was formed in loop 1 since it is an important binding site for antibody scFv PL-2. Proline locked the Spike-2-Oxford’s loop 1 in one conformation leading to the clashing of residues. The heavy chain of the scFv was clashing with residues of the loop 1 region and the Spike-2-Oxford protein was able to avoid being neutralized. A scFv was then engineered to have its heavy chain removed using site-directed mutagenesis. The scFv was expressed using the S2 insect cell system to bind the Spike-2-Oxford. The engineered scFv deemed to be “sticky” and so site-directed mutagenesis was once again performed but on Spike-2-Oxford to revert the proline to its wild type amino acid, serine. This was done to confirm if the amino acid change is indeed responsible for avoiding neutralization
Students
  • Edmundo Ismael Perez (Ten)
Mentors