Construction and characterization of an enhanced GFP-tagged TIM-1 fusion protein.
TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.
Department of Gynecology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, P.R. China.
Jilin
J
Qing
Haibing
H
Xiao
Lin
L
Zhao
Guifang
G
Qin
Lihua
L
Hu
Zhizhong
Z
Chen
D017209:Apoptosis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D000072596:Hepatitis A Virus Cellular Receptor 1; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C491143:HAVCR1 protein, human; D000072596:Hepatitis A Virus Cellular Receptor 1; D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins