Human Recombinant Resistin Protein Displays a Tendency To Aggregate by Forming Intermolecular Disulfide Linkages
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- 作者：Battu Aruna ; Sudip Ghosh ; Anil K. Singh ; Shekhar C. Mande ; V. Srinivas ; Radha Chauhan ; and Nasreen Z. Ehtesham
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：September 16, 2003
- 年：2003
- 卷：42
- 期：36
- 页码：10554 - 10559
- 全文大小：112K
- 年卷期：v.42,no.36(September 16, 2003)
- ISSN：1520-4995
文摘
Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a linkbetween obesity and type II diabetes by modulating the insulin signaling pathway and thus inducinginsulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at theamino acid level to any other known cysteine rich proteins. Resistin cDNA derived from humansubcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein.The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, aftersolubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysisof the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the proteinexhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography.The oligomeric structure was SDS-insensitive but 
chars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol-sensitive, pointing to the importanceof disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Classay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages.The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin,was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinantresistin has a tendency to reversibly convert from chars/alpha.gif" BORDER=0>-helical to chars/beta2.gif" BORDER=0 ALIGN="middle">-sheet structure as a direct function ofprotein concentration. Our novel observations on the biophysical and biochemical features of human resistin,particularly those shared with prion proteins, may have a bearing on its likely physiological function.
chars/beta2.gif" BORDER=0 ALIGN="middle">-mercaptoethanol-sensitive, pointing to the importanceof disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Classay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages.The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin,was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinantresistin has a tendency to reversibly convert from chars/alpha.gif" BORDER=0>-helical to chars/beta2.gif" BORDER=0 ALIGN="middle">-sheet structure as a direct function ofprotein concentration. Our novel observations on the biophysical and biochemical features of human resistin,particularly those shared with prion proteins, may have a bearing on its likely physiological function.