ABSTRACT
Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues. This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn. These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer.
Subject(s)
Carboxypeptidases/genetics , Carboxypeptidases/metabolism , Mutagenesis, Site-Directed , Alanine , Asparagine , Aspartic Acid , Carboxypeptidase B , Carboxypeptidases/chemistry , Escherichia coli/genetics , Gene Expression , Humans , Hydrolysis , Kinetics , Molecular Structure , Polymerase Chain Reaction , Structure-Activity Relationship , Substrate SpecificitySubject(s)
Antibodies, Monoclonal/genetics , Antibodies, Neoplasm/genetics , Escherichia coli/genetics , Genes, Immunoglobulin , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Peptide Fragments/biosynthesis , Protein Engineering/methods , Recombinant Fusion Proteins/biosynthesis , Amino Acid Sequence , Antibodies, Monoclonal/biosynthesis , Antibodies, Neoplasm/biosynthesis , Base Sequence , Cloning, Molecular , DNA/genetics , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Light Chains/biosynthesis , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunologyABSTRACT
A 511-base pair DNA fragment encoding human interferon-alpha 2 has been chemically synthesised and expressed from a lac UV5 and a synthetic trp promoter in Escherichia coli. The synthesis involved preparation of 68 oligodeoxyribonucleotides and their enzymic ligation. The product expressed from the trp promoter system had high antiviral activity and displayed biological effects similar to those of Namalwa interferon on natural killer cell activity and in a Daudi cell growth inhibition assay. E.coli minicells containing plasmid DNA with the synthetic IFN-alpha 2 gene under trp promoter control produce a protein with the same electrophoretic mobility as a sample of authentic IFN-alpha 2. The protein from E.coli cross-reacts with the monoclonal antibody NK-2 and was readily purified, close to homogeneity, by immunoadsorption chromatography on NK-2 sepharose.
Subject(s)
Cloning, Molecular , Escherichia coli/genetics , Interferon Type I/genetics , Amino Acid Sequence , Base Sequence , Biological Assay , Cell Line , DNA Replication/drug effects , DNA Restriction Enzymes , Humans , Interferon Type I/chemical synthesis , Interferon Type I/pharmacology , Kinetics , Nucleic Acid Hybridization , Oligonucleotides/chemical synthesis , PlasmidsABSTRACT
Cells of Escherichia coli containing a chemically synthesized human alpha 1 interferon (IFN-alpha 1) gene, under control of the lac promoter, make a product with biological properties indistinguishable from those of the natural IFN-alpha 1 [antiviral activity, acid stability, species crossreactivity, inactivation by antisera directed against leukocyte or Namalwa cell interferon, and stimulation of (2'-5')oligoadenylate synthetase activity]. Similar levels of IFN synthesis were obtained when the expression unit (lac promoter plus synthetic IFN-alpha 1 gene) was transplanted into the obligate methylotroph Methylophilus methylotrophus.
Subject(s)
Genes, Synthetic , Interferons/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , Biological Assay , DNA, Recombinant , Enzyme Activation/drug effects , Escherichia coli/genetics , Gene Expression Regulation , Humans , Hydrogen-Ion Concentration , Interferons/pharmacology , Methylococcaceae/genetics , PlasmidsABSTRACT
The concentration of prostaglandin F2alpha has been determined in serial samples of peripheral venous plasma from women at defined times during labour, and studied in detail throughout two consecutive uterine contractions. In addition, the same compound has been measured in single samples of uterine venous plasma, cord venous plasma, and amniotic fluid in groups of patients during early and late pregnancy, labour and at delivery of the baby.
