Location: Livestock Arthropod Pests Research
Project Number: 3094-32000-036-60-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Jul 31, 2019
Feasibility study: assess expression of R. microplus aquaporin 1 protein in N. benthamiana. (1) iBio CDMO proposes to test the expression of the R. micoplus aquaporin 1 protein. In discussion with USDA ARS, iBio designed antigen candidates to expression in plants. (2) USDA ARS will supply sequences from R. micoplus aquaporin 1 protein based on sequences from ARS patent # U.S.8,722,063 B2 dated May 13,2014.
1. iBio MB will codon optimize and order three codon variants of the Aquaporin 1 gene from a third party (2 weeks), 2. iBio MB will clone the candidate genes agreed to into iBio’s proprietary expression vectors, transform Agrobacterium tumefaciens GV3101, and confirm positive clones (2 weeks), 3. iBio MB will infiltrate plants to provide material for expression evaluation (2 weeks), 4. iBio PD will screen extraction conditions for the extraction of recombinant R. microplus Aquaporin 1 from infiltrated plant tissue (2 weeks), 5. If expression and extraction of Aquaporin 1 is successful, iBio PD will attempt the purification of Aquaporin 1 using StrepTrap HP resin (1 weeks), 6. iBio Analytical will perform basic protein characterization to confirm protein identity (SDS-PAGE, western blot, Mass Spectrometry), 7. If any purified material is generated from this initial purification run, it will be used for protein identification by mass spectrometry, positive controls, and deliverable to USDA ARS.