Expression Of Antifreeze Protein Gene From Two Tenebrodae Beetles In Xinjiang Desert And Characterization Of The Proteins

Antifreeze proteins (AFPs) were firstly identified by De Vries in 1969 in the blood of fish living in the polar Mcmurdo sea. Since then these proteins have been characterized from a variety of organisms such as plant, insect, microorganism, etc. Antifreeze proteins designate a class of proteins that are able to bind to ice and inhibit the generation and growth of ice crystal. AFPS can depress the freezeing point while not change the melting point of solution. These proteins can be used to preserve cells, tissues and organs from desiccating at low temperatures, thus AFPs could be used in the organ transplantation.The main tasks of this work are composed of five parts including expression of BkAFP972 in prokaryotic system, purification and characterization of BkAFP972, analysis of biological function of BkAFP972, physical-chemical characteristic of MpAFP5 which expressed in the yeast, and methodological studies on the detection of the antifreeze protein activity.1. Expression of BkAFP972 in prokaryotic system. BkAFP972 gene was cloned into pGEX-4T-1 prokaryotic expression vector, and the recombined plasmid was identified by sequence analysis and endonuclease digest analysis. The E.coli containing pGEX-4T-1- BkAFP972 was induced by IPTG and tested by SDS-PAGE. A 36KD molecular weight protein was produced, which is consistent with the deduced molecular weight of BkAFP972. The expression of BkAFP972 is important for its purification and preparation.2. Purification and characterization of BkAFP972. First, the GST-BkAFP972s were expressed at large scale and furtherly purified through Glutathione Sepharose 4B. The fusion...