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 r oles in collagen biosynthesis under normal and disease conditions. We descr ibe here a procedure for the expression and purification of Hsp47 in Escher ichia coli using the IMPACT expression system (New England Biolabs) where t he 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 com pared to other available protocols, especially when the bacterial cells wer e induced at 14 degreesC instead of 37 degreesC. The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from inte in using,beta -mercaptoethanol. Minor degradation products were subsequentl y removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies. Gel electrophoretic an alysis 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 sys tem allows for radiolabelling of purified Hsp47 with S-35 for use in bindin g experiments. Illustrative data on collagen binding by S-35-Hsp47 are show n. (C) 2001 Academic Press.