Preparation method of mulberry leaf metallothionein

Abstract

The invention discloses a preparation method of mulberry leaf metallothionein. The preparation method of the mulberry leaf metallothionein is completed through the six steps of mulberry leaf protein extracting, protein fusion vector expression constructing, biomembrane separating, orientated metal ion releasing, enzyme digestion eluting and purifying, refining and freeze-drying. A protein source of the mulberry leaf metallothionein is extracted and separated from pure plants, the plant protein resources are fully utilized, and compared with MT raw materials from other sources, the advantages of being reliable in raw material source, simple in extraction process, low in cost, green, safe and the like are achieved; when concentrated liquid protein is induced with metal ions, a metallic element which is necessary for a human body is selected as a fusion vector expression body, and therefore the target product can be stably expressed in a soluble and high-yield mode; the target protein and quantitative releasing of the metal ions can achieve classified expression to obtain purified MT protein molecules with the different structures; the extraction process cycle is short, the handling capacity is high, and the target product obtained through reacting has the advantages of being high in recycling rate, few in loss, high in purity, free of secondary pollution and the like.

Description

Technical field: [0001] The invention relates to a preparation method of mulberry leaf metallothionein, in particular to mulberry leaf protein, a production method for constructing protein fusion carrier expression and directional slow-release metal ion induced synthesis, belonging to the fields of biotechnology and protein engineering. Background technique: [0002] Metallothionein (MetalloThionein) is referred to as MT, and its chemical name is metallothiohistidine trimethyl inner salt. It is a class of low molecular weight (6000~7000 Daltons), rich in cysteine ​​( 20% to 30%), is one of the most important, most singular, most physiologically functional, sulfhydryl (-SH) active polypeptide substances found in organisms, does not contain histidine and aromatic amino acids; and can be induced by metals Metal-binding protein; known as "stress protein and kinesin" with special physiological functions, it plays a veritable role of "life housekeeper" in human metabolism and cell...

AI Technical Summary

Problems solved by technology

There are literatures showing that scientists have successively discovered and proved the existence of metallothionein with similar structure and basically the same physiological function in plants, microorganisms and marine organisms (Classification of Metallothionein, P.-A. Binz, J.H.R.Kgi, 1999); Plant metallothionein was first discovered by Casterline and Barnett from soybean roots in 1977; in 1987, Lane et al. discovered Ec protein from wheat embryos and proved that its amino acid sequence was consistent with animal MTs, thus proving that plants contain MTs; The plant metallothionein mentioned in the domestic patent of a preparation method of plant metallothionein (patent number: 200410044118) is a method of constructing plant metallothionein by genetic engineering technology and highly expressed in Escherichia coli. Instead of directly obtaining protein from pure plants; in addition, studies have shown that there are many reports on metallothionein-like (abbreviated as MT-like) in plants in China, such as tomato, bladderwort, physalis, spinach, and cuttlefish. The plant heavy metal binding proteins (peptides) isolated from the roots of the stockgrass belong to the class of MT, and there are no reports on companies that can really extract high-level MT proteins from pure plants and apply them to industrial production, while the previously reported molecular weight is about 10KD All of them belong to MT-like, and MT-like may be an artificial product in the extraction process, or due to impurity, small peptide polymerization, etc., so there are certain limitations for industrialization

Images