CLONING & EXPRESSION OF ANTI FREEZE PROTEIN FROM TENEBRIO MOLITOR

Abstract

Antifreeze proteins (AFPs) are a class of proteins that inhibit the growth of ice crystals in organisms that live in sub-zero environments. These proteins lower the freezing point of water in the body fluids of these organisms without changing the melting point, a phenomenon known as thermal hysteresis. Tmolitre refers to a specific species from which antifreeze proteins can be isolated. This species is often studied for its unique ability to survive in extremely cold environments, making its AFPs valuable for various applications, including cryopreservation and enhancing frost resistance in crops.The gene encoding the antifreeze protein is identified using bioinformatics tools and isolated from T. molitre DNA. This typically involves PCR amplification using specific primers designed for the AFP gene. The isolated AFP gene is inserted into a suitable cloning vector (such as pUC19 or pGEM-T) to create a recombinant DNA molecule. Restriction enzymes and ligase are used to facilitate the insertion.The recombinant vector is introduced into a host organism, commonly Escherichia coli (E. coli), via a process called transformation. The transformed E. coli cells are then cultured on selective media to identify those that have successfully incorporated the AFP gene.For protein expression, the AFP gene is sub-cloned into an expression vector (like pET series vectors) which has regulatory elements such as promoters and ribosome binding sites to drive the expression of the AFP gene. The host organism for expression is often a strain of E. coli engineered for high protein yield.The transformed E. coli is grown to a specific optical density, and protein expression is induced using an agent such as IPTG. .After induction, the cells are lysed, and the AFP is purified from the lysate using chromatographic techniques like affinity chromatography, which exploits a tag added to the protein for easy purification. AFPs are used to preserve cells, tissues, and organs at low temperatures by preventing ice crystal formation, which can damage biological samples.Introducing AFP genes into crops can enhance their frost resistance, thereby extending the growing season and reducing crop losses due to frost. AFPs are added to frozen food products to improve texture by inhibiting the recrystallization of ice during storage.Achieving high yields of functional AFPs can be challenging due to issues with protein folding and solubility in heterologous systems. It is crucial to ensure that the expressed AFP retains its antifreeze activity, which involves rigorous biochemical and biophysical characterization..The cloning and expression of antifreeze proteins from T. molitre represent a significant advancement in biotechnology with diverse applications. Continued research and development are essential to overcome existing challenges and maximize the benefits of AFPs.