ABSTRACT
We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.
Subject(s)
Antibodies, Monoclonal/metabolism , Diphtheria Toxin/metabolism , Immunoglobulin G/metabolism , Liposomes/metabolism , Recombinant Fusion Proteins/metabolism , Staphylococcal Protein A/metabolism , Animals , Cell Membrane Permeability/drug effects , Diphtheria Toxin/genetics , Escherichia coli/genetics , Hydrogen-Ion Concentration , Liposomes/immunology , Mice , Mutation , Rabbits , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Staphylococcal Protein A/geneticsABSTRACT
We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.
Subject(s)
Diphtheria Toxin/metabolism , Interleukin-2/metabolism , Interleukin-3/metabolism , Animals , Base Sequence , Binding Sites , Cell Membrane/metabolism , DNA Primers , Diphtheria Toxin/chemistry , Humans , Hydrogen-Ion Concentration , Liposomes , Mice , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.
Subject(s)
Diphtheria Toxin/chemistry , Interleukin-2/chemistry , Interleukin-3/chemistry , Receptors, Interleukin-3/drug effects , Animals , Cell Line , Cell Survival/drug effects , Diphtheria Toxin/metabolism , Diphtheria Toxin/toxicity , Interleukin-2/metabolism , Interleukin-2/toxicity , Interleukin-3/metabolism , Interleukin-3/toxicity , Mice , Protein Folding , Receptors, Interleukin-3/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicityABSTRACT
The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.
Subject(s)
Escherichia coli/enzymology , Peptide Synthases/biosynthesis , Alanine/metabolism , Chromatography, Thin Layer , Escherichia coli/genetics , Genes, Bacterial/genetics , Kinetics , Peptide Synthases/genetics , Plasmids , Substrate SpecificityABSTRACT
The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli was over-produced in strains harbouring recombinant plasmids bearing the murC gene under the control of the lac or trc promoter. Plasmid pAM1005, in which the promoter and ribosome-binding site region of murC were removed and in which the gene was directly under the control of promoter trc, led to a 2000-fold amplification of the L-alanine-adding activity after induction by isopropyl-thio-beta-D-galactopyranoside. The murC gene product was visualized as a 50-kDa protein accounting for approximately 50% of the cell protein. A two-step purification led to 1 g of a homogeneous protein from an 18-1 culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the murC gene. The presence of 2-mercaptoethanol and glycerol was essential for the stability of the enzyme. The Km values for UDP-N-acetylmuramic acid, L-alanine and ATP/Mg2+ were estimated at 100, 20 and 450 microM, respectively. Under the optimal in vitro conditions a turnover number of 928 min-1 was calculated and a copy number/cell of 600 could be roughly estimated. The specificity of the enzyme for its substrates was investigated with various analogues. The enzyme also catalysed the reverse reaction.
Subject(s)
Alanine/metabolism , Escherichia coli/enzymology , Peptide Synthases/isolation & purification , Uridine Diphosphate N-Acetylmuramic Acid/metabolism , Amino Acid Sequence , Base Sequence , Enzyme Stability , Molecular Sequence Data , Peptide Synthases/biosynthesis , Substrate SpecificityABSTRACT
An extract from Escherichia coli containing the L-alanine-adding enzyme with a high specific activity was prepared. Several compounds structurally related to L-alanine were tested as inhibitors of this activity. Intact amino and carboxyl groups were necessary for an interaction with the enzyme. Certain halogenated (haloalanines) or unsaturated (L-vinylglycine, L-propargylglycine, 3-cyano-L-alanine) amino acids were good inhibitors. Radioactive glycine, serine and 1-aminoethylphosphonic acid were tested as substrates. Whereas glycine or L-serine gave rise to the formation of the corresponding nucleotide product, no synthesis of UDP-N-acetylmuramyl-L-1-aminoethylphosphonic acid could be detected.
