Application of Engineered Carrot Antifreeze Protein in the Cryopreservation of Rice Cells by Adsorbing into Ice surface to Inhibit Recrystallization

The E. coli expressed carrot antifreeze protein (DcAFP) was purified by preparative HPLC, refolded by dialysis, and checked by analytical HPLC and SDS-PAGE. The gradient-concentrations of DcAFP in 3% glycerol were used to cryopreserve rice single cell by vitrification at -25degC, -75degC and -180degC. The result showed that DcAFP, at optimal concentration of 0.1 mg/ml, can visibly inhibit the liquid recrystallization of thawed rice cells. DcAFP treated rice cells had higher viability even than positive control and facilitated the efficient daughter cell division. The average diameter of inhibited ice crystals was about 1.0 mum that was far less than that of rice cell, and was the reason for the decrease of the rice cell damage. The result of molecular dynamics simulations suggested that DcAFP can adsorb into 1010 plane of ice crystal to form an imperfect surface complementarity, hence to inhibit the growth and recrystallization of ice granules. We modeled the interactions among rice cell, DcAFP and ice, and suggested that DcAFP can stack around the cell membrane by interaction. Therefore, the adsorbing of DcAFP to the ice surfaces can protect the rice cells from the damage of the ice crystals.