Production of linear polymers of HIV go120-binding domains

Smythe, Jason A.; Nardelli, Bernardetta; Chatterjee, Pramita; Gallo, Robert C.; Gershoni, Jonathan M.

doi:10.1093/protein/7.2.145

Cited by 5 publications

(3 citation statements)

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“…Other studies of tandem gene arrays have focused on increasing binding avidity (33), introducing a high concentration of antisense sequences (34), determining whether identical domains fold independently (35), and producing polymers to increase protein stability (36). In addition, genetic drift between domains may produce homologous but not identical sequences, which in turn may facilitate the ability of the domains to fold independently (35).…”

Section: Discussionmentioning

confidence: 99%

Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

Bradrick

Shattuck

Strader

et al. 1996

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) provides resistance to the antibacterial drug trimethoprim. This Rplasmid-encoded enzyme does not share any homology with chromosomal DHFR. A recent crystal structure of active, homotetrameric R67 DHFR (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018 -1025) indicates that a single active site pore traverses the length of the molecule. Since the center of the pore possesses exact 222 symmetry, site-directed mutagenesis of residues in the pore will produce four mutations/active site. To break this inevitable symmetry, four copies of the gene have been linked in frame to create an active monomer possessing the essential tertiary structure of native tetrameric R67 DHFR. The protein product, quadruple R67 DHFR, is 4 times the molecular mass of native R67 DHFR in SDSpolyacrylamide gel electrophoresis and is monomeric under nondenaturing conditions as measured by sedimentation equilibrium experiments. The catalytic activity of quadruple R67 DHFR is decreased only slightly when compared with native R67 DHFR. Folding of quadruple R67 DHFR is completely reversible at pH 5. However, at pH 8, folding is not fully reversible; this is likely due to a competition between productive intramolecular versus nonproductive intermolecular domain association. The production of a fully active, monomeric R67 DHFR variant will enable the design of more meaningful site-directed mutants where single substitutions per active site pore can be generated.Recent protein engineering studies of oligomeric proteins have attempted to shift the assembly equilibrium toward the protomer by the introduction of destabilizing mutations at subunit interfaces (Refs. 1-3 and references therein). These studies allow the functional role of protein-protein interactions at subunit interfaces to be probed. This strategy assumes that most oligomeric proteins possessing n protomers possess n active sites and is less useful for enzymes that have active sites to which several subunits contribute.In contrast to the above approach that forms monomers by dissociation of the native oligomeric species, we have constructed an active, stable monomeric form of R67 dihydrofolate reductase (DHFR) 1 by association. This strategy involves the use of genetic engineering techniques to covalently link four protomers together. This approach is necessarily different, since R67 DHFR is a homotetramer with a single active site pore, and stabilization of the 78-amino acid monomer would yield only a partial active site.In R67 DHFR, a 222 symmetry element occurs at the center of the pore, and crystallography and binding studies indicate that two molecules of either substrate (dihydrofolate/folate) or cofactor (NADPH) can bind to both sides of the active site pore under binary complex conditions (4, 5). Under ternary complex conditions, a total of two ligands can be bound; the possibilities include two NADPH molecules, two folate molecules, or one NADPH and one folate bound on opposite sides of the pore. T...

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Section: Discussionmentioning

confidence: 99%

Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

Bradrick

Shattuck

Strader

et al. 1996

Journal of Biological Chemistry

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“…For this, the GIL-encoding sequences (GIL inserts) were cloned into the asymmetric AvaI site (CTCGGG) found in the modified MBP expression vector pMalC-133-AvaI (see Materials and Methods). We previously demonstrated that cloning inserts into such AvaI sites drives the formation of tandem repeats of the inserts in the proper reading frame and orientation [15]. This is due to the fact that the internal asymmetry of the restriction site generates two different 5 0 overhangs, thereby forcing compatible inserts to clone in only one orientation.…”

Section: Production Of Gil-specific Polyclonal Serummentioning

confidence: 99%

A general insert label for peptide display on chimeric filamentous bacteriophages

Kaplan

Gershoni

2012

Analytical Biochemistry

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The foreign insert intended to be displayed via recombinant phage proteins can have a negative effect on protein expression and phage assembly. A typical example is the case of display of peptides longer than 6 amino acid residues on the major coat protein, protein VIII of the filamentous bacteriophages M13 and fd. A solution to this problem has been the use of "two-gene systems" generating chimeric phages that concomitantly express wild-type protein VIII along with recombinant protein VIII. Although the two-gene systems are much more permissive in regard to insert length and composition, some cases can still adversely affect phage assembly. Although these phages genotypically contain the desired DNA of the insert, they appear to be phenotypically wild type. To avoid false-negative results when using chimeric phages in binding studies, it is necessary to confirm that the observed lack of phage recognition is not due to faulty assembly and display of the intended insert. Here we describe a strategy for generating antibodies that specifically recognize recombinant protein VIII regardless of the nature of its foreign insert. These antibodies can be used as a general monitor of the display of recombinant protein VIII into phage particles.

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“…[191] Tabelle 5 enthält weitere Beispiele zur Zell-Zell-Wechselwirkung. [84] P: Transportprotein für Aminosäuren [85] Nichtökotropisches Mäuse-Leukämievirus Leukämie P: gp70 (Oberfläche) [86] HIV-1 AIDS P: gp120-ähnlicher Bereich der schweren Kette (Ig) P: gp120-ähnliches Neuroleukin P: gp120-ähnliches Vasoactive Intestinal Peptide [87,88] P: CD4 der T-Zellen P: CD4-Molekül wechselwirkt mit Klasse-II-HLA-DR-MHC Z: Galactosylceramid (oder nahe verwandtes Molekül auf epithelialen HT29-Zellen im menschlichen Dickdarm) P: CR2, besonders in Zellen, die mit EBV infiziert sind P: Chemokin-Rezeptoren CXCR-4 (T-Zell-trophisch) und CCR-5 (Makrophagen-trophisch) ± beide sind G-Protein-gekoppelte Rezeptoren mit sieben Transmembranhelices [89] HIV-2 AIDS P: CD4 [90] Simian Immunodeficiency Virus (SIV) Immunschwächesyndrom analog zu AIDS bei Affen P: SIV mac239 [91] P: CD4 Afrikanisches Schweinevirus P: p12, p72 [92,93] [a] P Protein, Z Zucker (Glycoprotein oder Glycolipid). [98] Z: Fucose-enthaltende Glycoside [99] auf Epitheloberfläche…”

Section: Bindung Von Zellen An Andere Zellen: Neutrophile Und Arterieunclassified

Polyvalente Wechselwirkungen in biologischen Systemen: Auswirkungen auf das Design und die Verwendung multivalenter Liganden und Inhibitoren

1998

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Überall in der Biologie kommen polyvalente Wechselwirkungen vor. Sie zeichnen sich durch die gleichzeitige Bindung mehrerer Liganden einer biologischen Einheit an mehrere Rezeptoren einer anderen biologischen Einheit aus (oberer Teil der Graphik) und haben eine Reihe von Charakteristika, die monovalenten Wechselwirkungen fehlen (unten). Besonders im Verbund können polyvalente Wechselwirkungen viel stärker sein als entsprechende monovalente Wechselwirkungen, und sie können die Basis für das Verständnis fördernder und hemmender biologischer Wechselwirkungen liefern, die sich grundsätzlich von denen in monovalenten Systemen unterscheiden.

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Production of linear polymers of HIV go120-binding domains

Cited by 5 publications

References 0 publications

Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

A general insert label for peptide display on chimeric filamentous bacteriophages

Polyvalente Wechselwirkungen in biologischen Systemen: Auswirkungen auf das Design und die Verwendung multivalenter Liganden und Inhibitoren

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