Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.
GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.
Zhuang
Ran
R
Department of Immunology, The Fourth Military Medical University, 17 Changle West Road, Xi'an 710032, PR China.
Zhang
Yuan
Y
Zhang
Rui
R
Song
Chaojun
C
Yang
Kun
K
Yang
Angang
A
Jin
Boquan
B
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D015696:Gene Products, tat / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins