RAPID PURIFICATION AND N-TERMINAL AMINO-ACID-SEQUENCE OF A PHOTOAFFINITY-LABELED JUVENILE-HORMONE BINDING-PROTEIN FROM AN ARCTIID MOTH LARVA, PLATYPREPIA-VIRGINALIS

被引：12

作者：

PRESTWICH, GD ^{[1
]}

ATKINSON, JK ^{[1
]}

机构：

[1] BODEGA BAY MARINE LAB,BODEGA BAY,CA 94923

来源：

INSECT BIOCHEMISTRY | 1990年 / 20卷 / 08期

基金：

美国国家科学基金会; 美国国家卫生研究院;

关键词：

JUVENILE HORMONE BINDING PROTEIN; PURIFICATION; LEPIDOPTERA ARCTIID LARVA; AMINO ACID SEQUENCE; ISOELECTRIC FOCUSING; PHOTOAFFINITY LABELING;

D O I：

10.1016/0020-1790(90)90098-F

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A novel two-step procedure has been developed for the purification of juvenile hormone binding proteins (JHBP) from caterpillars. Crude hemolymph was photoaffinity labeled with [H-3]EHDA, a JH II analog. After removal of excess ligand, 40 ml of buffer-diluted hemolymph containing over 200 mg protein was submitted to preparative isoelectric focusing (IEF) using a Rotofor device. After removal of ampholytes by dialysis, the H-3-labeled fractions were purified to > 95% homogeneity by anion-exchange HPLC. Over 1000-fold purification could be achieved in a few days on a scale which provides 100-1000-mu-g of purified JHBP. Proteins thus obtained can be used for proteolytic digestion or can be sequenced after electroblotting from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel onto a polyvinylidene fluoride (PVDF) membrane. This protocol is illustrated for the purification and N-terminal amino acid sequencing of a hemolymph JHBP from an arctiid wooly bear caterpillar, Platyprepia virginalis.

引用

页码：801 / 807

页数：7

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