LARGE-SCALE PURIFICATION, NUCLEOTIDE BINDING-PROPERTIES, AND ATPASE ACTIVITY OF THE MALK SUBUNIT OF SALMONELLA-TYPHIMURIUM MALTOSE TRANSPORT COMPLEX

Autor(en): WALTER, C
BENTRUP, KHZ
SCHNEIDER, E 
Stichwörter: BACTERIAL TRANSPORT; Biochemistry & Molecular Biology; CYSTIC-FIBROSIS; ESCHERICHIA-COLI; GENE; HISTIDINE PERMEASE; MEMBRANE-COMPONENTS; P-GLYCOPROTEIN; POLYACRYLAMIDE GELS; PROTEIN; SEQUENCES
Erscheinungsdatum: 1992
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 267
Ausgabe: 13
Startseite: 8863
Seitenende: 8869
Zusammenfassung: 
The malK gene, encoding a membrane-associated component of the maltose transport complex of Salmonella typhimurium was cloned into an expression vector downstream of the promoters lambda-p(R) and lambda-p(L) and a strong translation initiation region. Escherichia coli strain JM109 harboring the resulting plasmid pCW14 synthesized a protein of apparent molecular mass of 43 kDa upon temperature shift, as demonstrated by sodium dodecyl sulfate-gel electrophoresis. The identity of the protein was determined by N-terminal amino acid sequencing. The overproduced protein was sequestered in inclusion bodies as revealed by electron microscopy. The protein was purified to homogeneity on a large scale by disrupting the cells with a passage through a Ribi press, solubilizing the inclusion bodies with urea, and subsequent chromatography on Red Agarose. Purified MalK, as the membrane-bound MalK protein could be covalently modified by [gamma-P-32]8-azido-ATP. Furthermore, the purified protein bound [gamma-P-32] ATP with a dissociation constant of 150-mu-M and exhibited ATPase activity, which was stimulated by dimethyl-sulfoxide and inhibited by ADP.
ISSN: 00219258

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