Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion.
Decorin is a small leucine-rich proteoglycan which is a component of the extracellular matrix of many connective tissues. We have developed a method for expression and purification of decorin as a fusion protein in Escherichia coli. A PCR product coding for the 330-amino-acid-residue secreted form of bovine decorin core protein was cloned into the vector pMal-c for expression in E. coli as a maltose-binding protein (MBP) fusion. Expressed MBP-decorin tended to form insoluble aggregates resistant to degradation by E. coli intracellular proteases. Fusion protein was therefore solubilized from bacterial inclusion bodies using guanidine HCl and refolded by dilution of the chaotrope to minimally denaturing conditions and disulfide shuffling. Final purification included amylose resin affinity chromatography and size exclusion chromatography. The 79-kDa MBP-decorin fusion protein could be completely cleaved by factor Xa protease in 24 h to yield 43-kDa MBP and 36-kDa decorin core protein. The decorin core protein was separated from MBP and factor Xa protease by DEAE-Sephacel chromatography. Using a solid-phase assay, we have characterized its binding affinity for extracellular matrix components known to interact with tissue-derived decorin including collagen type VI, collagen type I, and fibronectin. The purification and refolding protocol described here may be generally applicable to bacterial expression of other members of this growing family of related small leucine-rich proteoglycan core proteins.
Hering
T M
TM
Department of Medicine, Case Western Reserve University, Cleveland, Ohio, 44106-4946, USA.
Kollar
J
J
Huynh
T D
TD
Varelas
J B
JB
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D002417:Cattle; D002850:Chromatography, Gel; D058575:Decorin; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D016326:Extracellular Matrix Proteins; D015951:Factor Xa / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D058575:Decorin; D029968:Escherichia coli Proteins; D016326:Extracellular Matrix Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D015951:Factor Xa