摘要
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.
出处
《西南农业大学学报(自然科学版)》
CAS
CSCD
1999年第3期209-214,共6页
Journal of Southwest Agricultural University
基金
国家自然科学基金
关键词
人雄激素受体
激素结合部
表达
优化
纯化
the human
androgen receptor (hAR)
the ligandbinding domain (LBD)
expression
optimization
Purification