Investigation of Antifreeze Proteins Applications for Cryopreservation of Biological Systems

Antifreeze proteins (AFPs) are produced as an adaptation by various organisms that inhabit subzero environments. AFPs have many potential applications in scientific research. However, a major bottleneck which hinders the extensive use of AFPs in research is the lack of sufficient availability and high cost. Our research focuses on large scale production of recombinant AFPs and investigation of their effect and underlying mechanism of action in cryopreservation.

Fed-batch fermentation technique is employed to obtain high cell density cultures of E. coli producing various recombinant AFPs (Type III AFP, GFP-AFP III, MBP-TmAFP and GFP-RiAFP). A unique protocol was developed to maximize expression yields that reached 1-2 g/L. The increase in productivity of fermentation process is 200 times higher comparing to shake flask conditions.

Production of such large amounts of AFPs enabled us to conduct further experiments with the aim of evaluating their potential application in cryopreservation of cells. We have examined the toxicity effect of Type III AFP and MBP-TmAFP on human epithelial colorectal adenocarcinoma cells (Caco-2) and immortal cell line derived from cervical cancer cells (HeLa). Without freezing, Type III AFP decreased cells viability at high concentrations (2.47, 1.23 and 0.62 mM), and had no toxic effect at concentration of 0.12 mM and lower. MBP-TmAFP at the concentration of 0.001-0.13 mM did not show cytotoxic effect. In cryopreservation, high concentrations of MBP-TmAFP (>30 μM) and Type III AFP (>10 μM) with 2% of DMSO decrease cell viability post thaw. Our preliminary results show that Type III AFP and MBP-TmAFP alone are not sufficient for cryopreservation of HeLa cells. Further, we intend to assess the effect of AFPs in vitrification.

Funding acknowledged by ISF and ERC.