酵母己糖激酶同工酶的纯化与结晶。通过色谱聚焦法对不同形式进行表征。

Purification and crystallization of yeast hexokinase isoenzymes. Characterization of different forms by chromatofocusing.

作者信息

Jacob L, Beecken V, Bartunik L J, Rose M, Bartunik H D

机构信息

Max-Planck-Society, Research Unit for Structural Molecular Biology, Hamburg, Germany.

出版信息

J Chromatogr. 1991 Nov 29;587(1):85-92. doi: 10.1016/0021-9673(91)85201-p.

DOI:10.1016/0021-9673(91)85201-p

PMID:1783664

Abstract

The yeast hexokinase isoenzymes PI and PII have been purified in large amounts (20 mg) from overproducing yeast strains. The purification procedures of hexokinase PI and PII include anion-exchange chromatography on DEAE-Sephacel and chromatofocusing on PBE 94, hydrophobic interaction chromatography on phenyl-Sepharose (necessary for the isolation of the isoenzyme PI); in the final step either a Mono Q HR 5/5 or a Fractogel EMD TMAE 650(S) column was used. Hexokinase preparations were characterized before crystallization by chromatofocusing on a Mono P HR 5/20 FPLC column, where different forms of hexokinase can be rapidly distinguished by their elution behaviour. From both purified hexokinase PI and PII, large crystals were grown that diffract X-rays to high resolution.

摘要

已从过量表达的酵母菌株中大量纯化（20毫克）了酵母己糖激酶同工酶PI和PII。己糖激酶PI和PII的纯化步骤包括在DEAE-琼脂糖凝胶上进行阴离子交换色谱以及在PBE 94上进行聚焦色谱，在苯基琼脂糖上进行疏水相互作用色谱（这是分离同工酶PI所必需的）；在最后一步中，使用了Mono Q HR 5/5或Fractogel EMD TMAE 650(S)柱。在结晶之前，通过在Mono P HR 5/20 FPLC柱上进行聚焦色谱对己糖激酶制剂进行表征，不同形式的己糖激酶可通过其洗脱行为快速区分。从纯化的己糖激酶PI和PII中都生长出了能将X射线衍射到高分辨率的大晶体。