Soluble expression and affinity purification of functional domain of human acetylcholine receptor alpha-subunit by the modulation of maltose binding protein.
An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.
Li
Zhuo-Yu
ZY
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, PR China.
Li
Yan-Jun
YJ
Guo
Chen-Yun
CY
Shi
Ye-Wei
YW
Xu
Ming-Qun
MQ
Trommer
Wolfgang E
WE
Yuan
Jing-Ming
JM
D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D058619:Maltose-Binding Proteins; D009363:Neoplasm Proteins / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D009363:Neoplasm Proteins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant