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A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone.

Abstract：

: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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Thomson CA,Ananthanarayanan VS

doi

10.1006/prep.2001.1470

keywords:

subject

Has Abstract

pub_date

2001-10-01 00:00:00

pages

8-13

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(01)91470-6

journal_volume

23

pub_type

杂志文章

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