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AnAFP

Introduction

Ammopiptanthus nanus antifreeze protein (AnAFP) is an antifreeze protein derived from Ammopiptanthus nanus. It protects the stability of membrane structures under cold conditions by binding to or dissociating from the membrane. It prevents the coagulation of macromolecules and plays a regulatory role by binding to or dissociating from calcium ions^[1].

Figure 1. The principle of antifreeze AnAFP

Usage and Biology

We heterologously expressed codon-optimized AnAFP in E.coli, endowing it with antifreeze capability.

Characterization

Sequencing Map

Figure 2. Sequencing map of AnAFP Sequencing starts from the T7 terminator, with the primer 5-GCTAGTTATTGCTCAGCGG-3.

Successful Protein Expression

Figure 3. SDS-PAGE electrophoresis of AnAFP We constructed AnAFP into the pET28a plasmid and transformed it into E. coli BL21 AI. Lanes 1 to 3 represent AnAFP, AnAFP + ara, and AnAFP + ara + IPTG, as indicated by the red arrow, we successfully expressed AnAFP.

AnAFP Antifreeze Assay

We tested the antifreeze capability of AnAFP and found that E. coli expressing AnAFP exhibited a higher survival rate under prolonged cold treatment conditions compared to E. coli transformed with an empty PET28 vector, indicating that it possesses antifreeze ability.

Figure 4. Workflow of AnAFP Antifreeze Assay

The experimental group consists of E. coli BL21 DE3 expressing AnAFP, the control group comprises E. coli BL21 DE3 transformed with the empty vector pET28a. All the groups are cultured overnight. After reaching an OD600 value of 1.0, they are subjected to cold treatment at 0 °C.Samples are taken at 0, 24, 48, 72 and 96 hours during cold treatment and are diluted 10^5 times in ddH2O for CFU counting. CFU counting is performed with the help of imageJ.

For each tube of cold-treated E. coli, the colony count at the measurement time is divided by the colony count at 0 hours to obtain the survival rate of E. coli in that tube at that specific time. The average survival rate for the same type of bacteria is then calculated, and survival curves for each group of E. coli are plotted.

Figure 5. Colonies at 0,24,48,72 and 96 hours of cold treatment at 0 °C From left to right is a time gradient of freezing treatment, while from up to down are the groups that were transformed with AnAFP and the control group.

Figure 3. Survival curves under cold treatment at 0 °C In the initial 48 hours, there was no significant difference in the survival rates between the experimental and control groups. However, after 48 hours, the survival rate of the experimental group was significantly higher than that of the control group.

Sequence and Features

References

1. ↑ Deng, L.-Q., Yu, H.-Q., Liu, Y.-P., Jiao, P.-P., Zhou, S.-F., Zhang, S.-Z., Li, W.-C., & Fu, F.-L. (2014). Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco. Gene, 539(1), 132-140.https://doi.org/10.1016/j.gene.2014.01.013