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The Use of poly(L-proline)-Sepharose in the Isolation of Profilin and Profilactin Complexes

The Use of poly(L-proline)-Sepharose in the Isolation of Profilin and Profilactin Complexes

U Lindberg, C E Schutt, E Hellsten, A C Tjäder, T Hult

Biochim Biophys Acta. 1988 Dec 15;967(3):391-400.

PMID: 3196757

Abstract:

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP25191133	Poly-L-proline		25191-13-3	Price

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