Cloning a codon-optimized version of the Rhagnium Inquisitor Antifreeze Protein gene into the pTYB12 expression vector

Abstract

The purpose of this project is to clone and express a synthetic version of antifreeze protein, RiAFP, from the arctic beetle, Rhagnium Inquisitor. The coding sequence for the RiAFP was codon-optimized for expression in E. Coli. Natively expressed RiAFP prevents the arctic beetle's body fluids from freezing by binding to the surface of ice crystals and inhibits the ability for the crystals to grow and expand.1 This protein exhibits a unique beta fold. The Beta sheets stack on top of each other in a repeating and parallel form. This protein is smaller than average as it has a size of 405 bp and a molecular weight of 13 kDA.2 It has been hypothesized that RiAFP may be applied to various cell-types in culture and improve cellular viability after the freezing process. So far, the codon-optimized version of the RiAFP gene has been successfully synthesized and amplified by PCR. The expected size of the amplicon, 405 bp, was verified with a 1 % agarose gel. After purification, the amplicon concentration was measured to be 263 ng/μL with a Qubit fluorimeter. The PCR product will be ligated into a pTYB12 expression vector backbone using HiFi assembly. Once successful cloning is confirmed via DNA sequencing the recombinant plasmid will be transformed into the BL21 expression strain of E. Coli. The RiAFP product will then be tested at various concentrations to test the hypothesis that inclusion in cryopreservation media will improve post thaw cellular viability. The data found from this project can hopefully be applied to the emerging field of cell therapy by investigating cellular freeze/thaw stress tolerance. 1.Lai Fun Kong, Ahmad Ayad Qatran Al-Khdhairawi, Bimo Ario Tejo; Rational design of short antifreeze peptides derived from Rhagium inquisitor antifreeze protein; Biocatalysis and Agricultural Biotechnology; Volume 23; 2020. 2.Hakim A, Nguyen JB, Basu K, Zhu DF, Thakral D, Davies PL, Isaacs FJ, Modis Y, Meng W; Crystal structure of an insect antifreeze protein and its implications for ice binding; J Biol Chem; Volume 17; 2013.