New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii.
Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to form oxalacetate. It is composed of three subunits, the central cylindrical hexameric 12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here report the cloning, sequencing, expression, and purification of the 5S subunit. The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database. For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a protein of 505 amino acids and of M(r) 55,700.
Kumar Bhat
Rakesh
R
Institute of Analytical Chemistry, University of Leipzig, Leipzig, Germany.
Berger
Stefan
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology / Q000235:genetics*; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001710:Biotin; D019289:Pyruvic Acid; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase