Tags

Type your tag names separated by a space and hit enter

cDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase.
Eur J Biochem. 1995 Jun 01; 230(2):533-7.EJ

Abstract

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme.

Links

  • Publisher Full Text

    • Authors+Show Affiliations

    Jagath-Reddy J
    Department of Biochemistry, Indian Institute of Science, Bangalore.
    Ganesan K
    No affiliation info available
    Savithri HS
    No affiliation info available
    Datta A
    No affiliation info available
    Rao NA
    No affiliation info available

    MeSH

    Amino Acid SequenceAnimalsBase SequenceCloning, MolecularCytosolDNA, ComplementaryEscherichia coliGlycine HydroxymethyltransferaseLiverMolecular Sequence DataSheep

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    7607226

    Citation

    Jagath-Reddy, J, et al. "CDNA Cloning, Overexpression in Escherichia Coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase." European Journal of Biochemistry, vol. 230, no. 2, 1995, pp. 533-7.
    Jagath-Reddy J, Ganesan K, Savithri HS, et al. CDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase. Eur J Biochem. 1995;230(2):533-7.
    Jagath-Reddy, J., Ganesan, K., Savithri, H. S., Datta, A., & Rao, N. A. (1995). CDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase. European Journal of Biochemistry, 230(2), 533-7.
    Jagath-Reddy J, et al. CDNA Cloning, Overexpression in Escherichia Coli, Purification and Characterization of Sheep Liver Cytosolic Serine Hydroxymethyltransferase. Eur J Biochem. 1995 Jun 1;230(2):533-7. PubMed PMID: 7607226.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - cDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase. AU - Jagath-Reddy,J, AU - Ganesan,K, AU - Savithri,H S, AU - Datta,A, AU - Rao,N A, PY - 1995/6/1/pubmed PY - 1995/6/1/medline PY - 1995/6/1/entrez SP - 533 EP - 7 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 230 IS - 2 N2 - A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/7607226/cDNA_cloning_overexpression_in_Escherichia_coli_purification_and_characterization_of_sheep_liver_cytosolic_serine_hydroxymethyltransferase_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=1995&volume=230&issue=2&spage=533 DB - PRIME DP - Unbound Medicine ER -