An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies

被引:7
作者
Aatsinki, JT
Rajaniemi, HJ
机构
[1] Univ Oulu, Inst Dent, FIN-90014 Oulu, Finland
[2] Univ Oulu, Dept Anat & Cell Biol, FIN-90014 Oulu, Finland
关键词
affinity purification; antibody production; fusion protein; glutathione S-transferase; luteinizing hormone/chorionic gonadotropin receptor;
D O I
10.1016/j.pep.2004.11.012
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor. (c) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:287 / 291
页数:5
相关论文
共 17 条
[1]  
Aatsinki J T, 1997, Methods Mol Biol, V67, P55
[2]  
AATSINKI JT, 1994, BIOTECHNIQUES, V16, P282
[3]   EXPRESSION OF THE LH/CG RECEPTOR GENE IN RAT OVARIAN TISSUE IS REGULATED BY AN EXTENSIVE ALTERNATIVE SPLICING OF THE PRIMARY TRANSCRIPT [J].
AATSINKI, JT ;
PIETILA, EM ;
LAKKAKORPI, JT ;
RAJANIEMI, HJ .
MOLECULAR AND CELLULAR ENDOCRINOLOGY, 1992, 84 (1-2) :127-135
[4]  
AATSINKI JT, 1998, BIOTECHNIQUES UPDATE, P261
[5]  
Aatsinki Jyrki T, 2002, Methods Mol Biol, V192, P53
[6]   Identification and structural characterization of the neuronal luteinizing hormone receptor associated with sensory systems [J].
Apaja, PM ;
Harju, KT ;
Aatsinki, JT ;
Petäjä-Repo, UE ;
Rajaniemi, HJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2004, 279 (03) :1899-1906
[7]   A method for isolation and purification of specific antibodies to a protein fused to the GST [J].
BarPeled, M ;
Raikhel, NV .
ANALYTICAL BIOCHEMISTRY, 1996, 241 (01) :140-142
[8]   An improved dual-expression concept, generating high-quality antibodies for proteomics research [J].
Falk, R ;
Agaton, C ;
Kiesler, E ;
Jin, S ;
Wieslander, L ;
Visa, N ;
Hober, S ;
Ståhl, S .
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, 2003, 38 :231-239
[9]  
GRIECO F, 1992, BIOTECHNIQUES, V13, P856
[10]  
KEINANEN KP, 1987, J BIOL CHEM, V262, P7920