THE 24 KDA OUTER ENVELOPE MEMBRANE-PROTEIN FROM SPINACH-CHLOROPLASTS - MOLECULAR-CLONING, IN-VIVO EXPRESSION AND IMPORT PATHWAY OF A PROTEIN WITH UNUSUAL PROPERTIES

被引:44
作者
FISCHER, K
WEBER, A
ARBINGER, B
BRINK, S
ECKERSKORN, C
FLUGGE, UI
机构
[1] UNIV WURZBURG, JULIUS VON SACHS INST BIOWISSENSCH, D-97082 WURZBURG, GERMANY
[2] MAX PLANCK INST BIOCHEM, D-82152 MARTINSRIED, GERMANY
关键词
SPINACIA OLERACEA; CHEMICAL CLEAVAGE; GENE EXPRESSION; POLYMERASE CHAIN REACTION; PROTEIN TRANSPORT; SDS-PAGE;
D O I
10.1007/BF00023235
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
The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.
引用
收藏
页码:167 / 177
页数:11
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