Development of High Throughput Screening for Variable Lymphocyte Receptors (VLRs) Using E. coli Expression System

Abstract

Variable lymphocyte receptors (VLRs) of jawless vertebrates are the only natural adaptive immune system except the immunoglobulin (Ig) fold of jawed vertebrates. VLR contains up to 9 leucine-rich repeat (LRR) modules that form a right-handed horseshoe-shaped solenoidal assembly. The assembled LRR modules increase diversity of the concave surface that recognizes specific antigens, which accomplish the comparable repertoire up to 1014 as many as antibodies. This suggests the probability of VLRs as antibody alternatives. VLRs had a difficulty in soluble expression using E. coli system, so I focused on the development of the VLR expression and purification system using E. coli system in a high throughput manner. I carried out experimental steps using some bioengineering techniques including an InlB-fusion construct generation, which enable to succeed in mass-production of soluble and stable VLRs. I established the methods of high-throughput screening for VLRs that have high affinity to specific antigens. Meanwhile, crystallization was done with a randomly chosen VLR protein. Here, the crystal structure was determined at 1.95 Å resolution.