scholarly journals Purification and characterization of human H-ras proteins expressed in Escherichia coli

1985 ◽  
Vol 5 (5) ◽  
pp. 1015-1024
Author(s):  
M Gross ◽  
R W Sweet ◽  
G Sathe ◽  
S Yokoyama ◽  
O Fasano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Activity ◽  
Full Length ◽  
Binding Kinetics ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

scholarly journals Purification and characterization of human H-ras proteins expressed in Escherichia coli.

1985 ◽  
Vol 5 (5) ◽  
pp. 1015-1024 ◽  
Author(s):  
M Gross ◽  
R W Sweet ◽  
G Sathe ◽  
S Yokoyama ◽  
O Fasano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Activity ◽  
Full Length ◽  
Binding Kinetics ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

scholarly journals Identification of Epitope and Surface-Exposed Domains of Shigella flexneri Invasion Plasmid Antigen D (IpaD)

1998 ◽  
Vol 66 (5) ◽  
pp. 1999-2006 ◽  
Author(s):  
K. Ross Turbyfill ◽  
Jennifer A. Mertz ◽  
Corey P. Mallett ◽  
Edwin V. Oaks
Keyword(s):  
Amino Acid ◽  
Shigella Flexneri ◽  
Serum Antibody ◽  
Terminal Region ◽  
Virulence Plasmid ◽  
Amino Acid Residues ◽  
Unique Region ◽  
Amino Terminal ◽  
Convalescent Phase ◽  
Carboxyl Terminal

ABSTRACT Transport and surface expression of the invasion plasmid antigens (Ipa proteins) is an essential trait in the pathogenicity ofShigella spp. In addition to the type III protein secretion system encoded by the mxi/spa loci on the large virulence plasmid, transport of IpaB and IpaC into the surrounding medium is modulated by IpaD. To characterize the structural topography of IpaD, the Geysen epitope-mapping system was used to identify epitopes recognized by surface-reactive monoclonal and polyclonal antibodies produced against purified recombinant IpaD or synthetic IpaD peptides. Surface-exposed epitopes of IpaD were confined to the first 180 amino acid residues, whereas epitopes in the carboxyl-terminal half were not exposed on the Shigella surface. By using convalescent-phase sera from 10 Shigella flexneri-infected monkeys, numerous epitopes were mapped within a surface-exposed region of IpaD between amino acid residues 14 and 77. Epitopes were also identified in the carboxyl-terminal half of IpaD with a few convalescent-phase sera. Comparison of IpaD epitope sequences withSalmonella SipD sequences indicated that very similar epitopes may exist in the carboxyl-terminal region of each protein whereas the IpaD epitopes in the surface-exposed amino-terminal region were unique for the Shigella protein. Although the IpaD and SipD homologs may play similar roles in transport, the dominant serum antibody response to IpaD is against the unique region of this protein exposed on the surface of the pathogen.

Using Small Peptide Segments of Amyloid-β and Humanin to Examine their Physical Interactions

2019 ◽  
Vol 26 (7) ◽  
pp. 502-511 ◽  
Author(s):  
Deborah L. Heyl ◽  
Brandon Iwaniec ◽  
Daniel Esckilsen ◽  
Deanna Price ◽  
Prathyusha Guttikonda ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Full Length ◽  
Binding Kinetics ◽  
Amino Acid Residues ◽  
Aβ Peptides ◽  
Physical Interactions ◽  
Peptide Segments ◽  
Kinetics Of

Background: Amyloid fibrils in Alzheimer’s disease are composed of amyloid-β (Aβ) peptides of variant lengths. Humanin (HN), a 24 amino acid residue neuroprotective peptide, is known to interact with the predominant Aβ isoform in the brain, Aβ (1-40). Methods: Here, we constructed smaller segments of Aβ and HN and identified residues in HN important for both HN-HN and HN-Aβ interactions. Peptides corresponding to amino acid residues 5- 15 of HN, HN (5-15), HN (5-15, L11S), where Leu11 was replaced with Ser, and residues 17-28 of Aβ, Aβ (17-28), were synthesized and tested for their ability to block formation of the complex between HN and Aβ (1-40). Results: Co-immunoprecipitation and binding kinetics showed that HN (5-15) was more efficient at blocking the complex between HN and Aβ (1-40) than either HN (5-15, L11S) or Aβ (17-28). Binding kinetics of these smaller peptides with either full-length HN or Aβ (1-40) showed that HN (5- 15) was able to bind either Aβ (1-40) or HN more efficiently than HN (5-15, L11S) or Aβ (17-28). Compared to full-length HN, however, HN (5-15) bound Aβ (1-40) with a weaker affinity suggesting that while HN (5-15) binds Aβ, other residues in the full length HN peptide are necessary for maximum interactions. Conclusion: L11 was more important for interactions with Aβ (1-40) than with HN. Aβ (17-28) was relatively ineffective at binding to either Aβ (1-40) or HN. Moreover, HN, and the smaller HN (5-15), HN (5-15 L11S), and Aβ (17-28) peptides, had different effects on regulating Aβ (1-40) aggregation kinetics.

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 ◽  
Vol 67 (05) ◽  
pp. 526-532 ◽  
Author(s):  
Glenn T G Chang ◽  
Hans K Ploos van Amstel ◽  
Martin Hessing ◽  
Pieter H Reitsma ◽  
Rogier M Bertina ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Recombinant Protein ◽  
Plasma Protein ◽  
Binding Protein ◽  
Protein S ◽  
Full Length ◽  
Human Protein ◽  
Amino Acid Residues ◽  
Amino Terminal

SummaryMouse C127 epithelioid cells were genetically engineered to produce biologically active ³-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 ³-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 ³-carboxylated and fully p-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 gin, respectively and this variant was designated C variant. The binding of these variants to C4BP was the same as wild type recombinant protein S. This suggests that amino acid residues leu-608, asp-609, asp-611 and glu-612 are not essential for binding of the intact full length protein to C4BP.

scholarly journals Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

2007 ◽  
Vol 405 (3) ◽  
pp. 465-472 ◽  
Author(s):  
Elina Jakobsson ◽  
Anne Jokilammi ◽  
Juha Aalto ◽  
Pauli Ollikka ◽  
Jukka V. Lehtonen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Structural Model ◽  
Polysialic Acid ◽  
Binding Activity ◽  
Enzymic Activity ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Escherichia Coli K1

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of ‘host range’ mutants of E. coli K1 bacteriophages.

scholarly journals DNA-Binding Activity of Amino-Terminal Domains of the Bacillus subtilis AbrB Protein

2001 ◽  
Vol 183 (13) ◽  
pp. 4094-4098 ◽  
Author(s):  
Ke Xu ◽  
Mark A. Strauch
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Disulfide Bonding ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Binding Affinity ◽  
Truncated Variants

ABSTRACT Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified. Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity. Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB. These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

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