A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone.
Hsp47 is regarded as a collagen-specific chaperone with several suggested roles in collagen biosynthesis under normal and disease conditions. We describe here a procedure for the expression and purification of Hsp47 in Escherichia coli using the IMPACT expression system (New England Biolabs) where the guest gene is fused to the adduct, intein, with a chitin-binding domain. Use of this system resulted in relatively high levels of soluble Hsp47 compared to other available protocols, especially when the bacterial cells were induced at 14 degrees C instead of 37 degrees C. The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using beta-mercaptoethanol. Minor degradation products were subsequently removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies. Gel electrophoretic analysis of the purified protein indicated the presence of a small proportion of trimeric species when non-reducing conditions were used. The ability to form a trimer may be important for its role as a chaperone. The IMPACT system allows for radiolabelling of purified Hsp47 with (35)S for use in binding experiments. Illustrative data on collagen binding by (35)S-Hsp47 are shown.
Thomson
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Department of Biochemistry, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada.
Ananthanarayanan
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D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D024042:Collagen Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D051381:Rats; D013462:Sulfur Radioisotopes; D013696:Temperature
D024042:Collagen Type I; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C494662:Serpinh1 protein, rat; D013462:Sulfur Radioisotopes