人雄激素受体LBD基因高效表达条件的优化及产物纯化

A STUDY ON OVERPRODUCTION OF LBD OF HAR IN E.coli AND PRODUCT PURIFICATION

人雄激素受体（ｈＡＲ）激素结合部（ＬＢＤ）的ｃＤＮＡ片段克隆到表达质粒ｐＴｒｘＡＲ内，在大肠杆菌ＧＩ７２４中可被诱导表达。通过改变诱导培养时间、诱导剂浓度、诱导温度及培养基的ｐＨ，在３４℃～３７℃，ｐＨ６．４～７．７的培养基中，用１００μｇ／ｍｌＴｒｐ诱导培养４ｈ，可获得高效表达的ＬＢＤ融合蛋白产物。其最高表达量为５０～７０ｍｇ／Ｌ湿菌体。产物主要以包含体形式存在。经尿素溶解后，由ＳｅｐｈａｃｒｙｌＳ－２００柱层析获得了电泳纯的目的产物。氨基酸组分分析和相对分子量测定表明与理论值一致。该表达条件的确定，为ｈＡＲ的ＬＢＤ基因表达和进一步研究提供了依据。

pTrxAR was constructed by cloning hAR's LBD gene fragment into Cterminal of Thioredoxin gene at pTrxFus. LBD gene fragment was expressed within Thioredoxin Fusion Expression System. By inducing, the target gene was expressed, and the target protein was detected with approximate expected molecular weight (46.56 KD). By changing the inducing time, inducer concentration, inducing temperature and pH of medium, it was suggested that satisfied expession could be obtained under 3437, 100 g/ml Trp, pH 6.47.7 and 4 h inducing time, and the yield of target protein reaches 5070 mg/L bacteria.The LBD gene of hAR was expressed as a form of inclusion bodies. It can not be purified by osmotic shock. The rough inclusion bodies sample was gained by lysing the harvested bacteria, centrifuging the lysate and washing the pellets. The target protoin was purified to homogeneity by solubilzation of inclusion bodies and gel friltration (Sephacryl S200). The constituent of amino acids and molecular weight of target protein are consistent to that of the expectaton.