Methods in Biosynthesis and Characterization of the Antifreeze Protein (AFP) for Potential Blood Cryopreservation

The cryopreservation of red blood cells (RBCs) is very important to modern medicine. Cryoprotectants (CPAs) such as dimethyl sulfoxide (DMSO), proline, trehalose, and polyvinyl alcohol (PVA) have been used in the cryopreservation of RBCs, but the results are not satisfactory. Marinomonas primoryensis antifreeze protein (MpAFP) is a Ca2+-dependent AFP derived from Antarctic bacteria, which can prevent bacteria from freezing under extremely cold conditions and may be suitable for cryopreservation of RBCs. The active region of MpAFP is located in region IV and is called MPAFP_RIV. In this paper, the gene of region IV of MpAFP is introduced into BL21 (DE3) competent cells, and MpAFP_RIV is obtained after culture, separation, and purification. The improved splat assay is proposed, and this method first proves that MpAFP_RIV has strong ice recrystallization inhibition (IRI) activity without Ca2+. The improved splat assay is easier to operate and lower the cost compared to the traditional one, and the results are consistent with the classic sucrose sandwich assay, proving that this method can accurately detect IRI activity. The feasibility of MPAFP_RIV combined with classical CPA for cryopreservation of RBCs and the methods to increase the yield of MPAFP_RIV are proposed.