| Autor: |
Thomson, Christy A., Ananthanarayanan, Vettai S. |
| Zdroj: |
Protein Expression and Purification; October 2001, Vol. 23 Issue: 1 p8-13, 6p |
| Abstrakt: |
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 coliusing 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°C instead of 37°C. The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using β-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 35S for use in binding experiments. Illustrative data on collagen binding by 35S-Hsp47 are shown. |
| Databáze: |
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