Recombinant Expression Of Tartary Buckwheat Tbw16Allergenand Preliminary Analysis Of Its Targeting Binding Protein

Buckwheat has balanced ratio of amino acids and abundant flavonoid. Because of its short growthduration, high resistance, leanness tolerance and other characteristics, buckwheat is an important food cropof relief supplies in China and other places around the world. Moreover, buckwheat can be used asfunctional food for prevention hyperglycemia, hyperlipidemia, hypertension, cancer, liver cirrhosis andother diseases. However, researchers found that some proteins in buckwheat could arouse unfavorablereaction, buckwheat allergy, which could harm the human health and limit the utilization of buckwheatfood.In this study, based on the tartary buckwheat16kD allergen (TBW16), which is located in seedembryo, prokaryotic expression vector pET47b-TBW16was constructed. On the basis of purificationTBW16, the interacting protein of TBW16was separated by affinity chromatography. This laid the basisfor revealing the biological function of TBW16and main results of this research were as followed:Based on the recombinant plasmid pTriplEx2-TBW16, the expression vector pET47b-TBW16ofmature protein deleting N-terminal signal peptide sequence (MKLFIILATATLLIAATQA) was successfullyconstructed, and then transferred it into BL21Star (DE3). The result showed that recombinant protein wassuccessfully overexpressed in E.coli BL21Star (DE3) in form of inclusion bodies. After renaturation bydialysis against gradually decreasing urea solution, TBW16was further purified by cobalt chelatingchromatography.TBW16was then coupled to Sepharose CL6B activated by1,4butyl glycol two glycidyl ether, andthe TBW16interacting protein was obtained by affinity chromatography protocols. After separatinginteracting protein with0.1%SDS and SDS-PAGE, the result showed that there was a clear about40kDband. Result of MALDI-TOF mass spectrometry showed that three peptides of TBW16interacting proteinhave high homology to bacteria porin. This result laid the basis for further revealing the biologicalfunctions of TBW16in Tartary buckwheat.