Acyl carrier protein of Azospirillum brasilense: properties of the purified protein and sequencing of the corresponding gene, acpP.
Acyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in Escherichia coli is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene (acpP) from Azospirillum brasilense using conventional methods. Therefore, ACP from A. brasilense was purified to homogeneity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the acpP region generated by two oppositely oriented internal primers designed from the partial acpP gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2.0-3.0 x 10(4) molecules per cell), (b) compared to E. coli ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.
Maiti
M K
MK
Department of Biochemistry, Bose Institute, Calcutta, India.
Ghosh
S
S
D000213:Acyl Carrier Protein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D015806:Azospirillum brasilense / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000213:Acyl Carrier Protein; D017931:DNA Primers; D011994:Recombinant Proteins