BIOMAT Database Logo BIOMAT Database Logo

Expression and characterization of recombinant human protein S in heterologous cells--studies of the interaction of amino acid residues leu-608 to glu-612 with human C4b-binding protein. 1992

G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Department of Haematology, University Hospital Utrecht, The Netherlands.

Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)

UI MeSH Term Description Entries
D007930 Leucine An essential branched-chain amino acid important for hemoglobin formation. L-Leucine,Leucine, L-Isomer,L-Isomer Leucine,Leucine, L Isomer
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D001798 Blood Proteins Proteins that are present in blood serum, including SERUM ALBUMIN; BLOOD COAGULATION FACTORS; and many other types of proteins. Blood Protein,Plasma Protein,Plasma Proteins,Serum Protein,Serum Proteins,Protein, Blood,Protein, Plasma,Protein, Serum,Proteins, Blood,Proteins, Plasma,Proteins, Serum
D002352 Carrier Proteins Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes. Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D003169 Complement Inactivator Proteins Serum proteins that negatively regulate the cascade process of COMPLEMENT ACTIVATION. Uncontrolled complement activation and resulting cell lysis is potentially dangerous for the host. The complement system is tightly regulated by inactivators that accelerate the decay of intermediates and certain cell surface receptors. Complement Cytolysis Inhibiting Proteins,Complement Cytolysis Inhibitor Proteins,Complement Inactivating Proteins,Serum Complement Inactivators,Complement Inactivators, Serum,Inactivating Proteins, Complement,Inactivator Proteins, Complement,Inactivators, Serum Complement,Proteins, Complement Inactivating,Proteins, Complement Inactivator
D004251 DNA Transposable Elements Discrete segments of DNA which can excise and reintegrate to another site in the genome. Most are inactive, i.e., have not been found to exist outside the integrated state. DNA transposable elements include bacterial IS (insertion sequence) elements, Tn elements, the maize controlling elements Ac and Ds, Drosophila P, gypsy, and pogo elements, the human Tigger elements and the Tc and mariner elements which are found throughout the animal kingdom. DNA Insertion Elements,DNA Transposons,IS Elements,Insertion Sequence Elements,Tn Elements,Transposable Elements,Elements, Insertion Sequence,Sequence Elements, Insertion,DNA Insertion Element,DNA Transposable Element,DNA Transposon,Element, DNA Insertion,Element, DNA Transposable,Element, IS,Element, Insertion Sequence,Element, Tn,Element, Transposable,Elements, DNA Insertion,Elements, DNA Transposable,Elements, IS,Elements, Tn,Elements, Transposable,IS Element,Insertion Element, DNA,Insertion Elements, DNA,Insertion Sequence Element,Sequence Element, Insertion,Tn Element,Transposable Element,Transposable Element, DNA,Transposable Elements, DNA,Transposon, DNA,Transposons, DNA
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle

Related Publications

G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Involvement of amino acid residues 423-429 of human protein S in binding to C4b-binding protein.
June 1998, Blood cells, molecules & diseases,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Commentary: Involvement of Amino Acid Residues 423-429 of Human Protein S in Binding to C4b-Binding Protein.
June 1998, Blood cells, molecules & diseases,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Characterization of the interaction between human protein S and C4b-binding protein (C4bp).
September 1987, Journal of biochemistry,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Solution-phase equilibrium binding interaction of human protein S with C4b-binding protein.
March 1991, Biochemistry,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C.
January 1995, The Biochemical journal,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Protein S enhances C4b binding protein interaction with neutrophils.
January 1993, Blood,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein.
November 1991, The Journal of biological chemistry,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Characterization of the interaction of human C4b-binding protein with physiological ligands.
November 1984, The Journal of biological chemistry,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
Expression and characterization of a recombinant C4b-binding protein lacking the beta-chain.
June 1995, The Biochemical journal,
G T Chang, and H K Ploos van Amstel, and M Hessing, and P H Reitsma, and R M Bertina, and B N Bouma
The C4b-binding protein-protein S interaction is hydrophobic in nature.
October 1998, Biochimica et biophysica acta,
Copied contents to your clipboard!