Antifreeze proteins (AFPs) can bind to forming ice crystals and prevent or limit their growth, reducing the damage they cause to living organisms. Due to this capability, they have numerous potential biotechnological applications in fields such as agriculture, cryopreservation, or material engineering (Eskandari et al., 2020).
AFP have the ability to both avoid the ice crystals formation and to control their shape and size. They do this by the gibbs-thompson effect, attaching to the forming ice nucleus and increasing the interface area, reducing the stability of the crystal. This causes a separation between the freezing point and the melting point that's called thermal hysteresis.
Taken from Ghazaleh and coworkers (Gharib et al ., 2022)
Despite their amazing properties, there is a great knowledge gap regarding their characteristics and possible applications. The first examples of AFPs were found in fish during the 70´s; however, as ocean water freezes at approximately -1,8°C, fish antifreeze proteins produce a maximum thermal hysteresis of approximately this temperature. Because of this, many application have been limited.
Here I propose to design a new protocol for producing hyperactive AFPs from Dendroides canadensis that have three times the thermal hysteresis activity that fishes possess. Once the protein is obtained, my aim is to use to make antifreeze materials and AFPs slow release systems.
To begin with the project I did an exhaustive bibliographic search to find the best AFPs candidates. The AFP from the desert beetle D.canadiensis is a particularly good candidate, it maintains its activity at temperatures as high as 100°C ,which makes it posible to store it easily and use it in coating processes that reach high temperatures. It has one of the most powerful thermal hysteresis activities of up to 6°C, meaning freezing is spontaneous at -6°C.
The insect produces different isoforms of this protein, which have been previously demonstrated to interact physically and to enhance each others activities (Wang et al., 2005). This two isoforms DAFP1 and DAFP 2 are two very similar proteins that differ only in two amino acids. To facilitate the interaction and enhancement outside the the insects physiological conditions, I designed a fusion protein with both isoforms linked with a linker peptide (Kong et al., 2016)that can be expressed in a single translation unit. The construction with the isoforms and the peptide were modeled in alphafold 3 and the following structure was obtained:
The different domains of the protein have internal disulfide bridges that stabilize the structure. As the bacterial cytoplasm has a high reducing potential, the formation of this bridges is quite difficult to achieve. Because of this, yeast expression systems are a better option to form this protein. So I optimize the nucleotides sequence for expression in Saccharomyces cerevisiae and insert it in a yeast episomal plasmid (pRS420) under an inducible promoter (CYC1).
But things really get interesting when it comes to the purification step. As AFPs have the unique property of attaching to ice crystals, this can be used in the purification step. The cold finger method allows to use this property to purificate the AFPs from a complex solution by introducing a cold finger coated with a thin ice layer. Once the layer is introducing the circulating sub-zero circulating liquid grows the ice outside the finger, and AFPs stay trapped in the frozen portion of the solution.