Selection of β-Lactamase on Filamentous Bacteriophage by Catalytic Activity

Soumillion, Patrice; Jespers, Laurent; Bouchet, Michèle; Marchand‐Brynaert, Jacqueline; Winter, Greg; Fastrez, Jacques

doi:10.1006/jmbi.1994.1244

Cited by 126 publications

(72 citation statements)

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“…This method of rapidly screening large protein libraries for rare variants has been applied to diverse uses such as the selection of linkers for catalytic antibodies (61) and the selection of enzymes based on substrate affinity (21,22). In this study we describe the successful adaptation of phage display methods to include the selection of metalloenzyme variants based on metal affinity.…”

Section: Discussionmentioning

confidence: 99%

Selection of Carbonic Anhydrase Variants Displayed on Phage

Hunt¹,

Fierke²

1997

Journal of Biological Chemistry

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In all metalloenzymes, hydrophobic residues surround the metal binding site. In carbonic anhydrase II (CAII) residues Phe 93 , Phe 95 , and Trp 97 flank two of the three histidines that coordinate zinc to form a hydrophobic cluster beneath the zinc binding site. A library of CAII variants differing in these hydrophobic amino acids was prepared using cassette mutagenesis, then displayed on filamentous phage, and screened for proteins retaining high zinc affinity. Wild-type CAII was enriched 20-fold by selection, and consensus residues at each position were identified from the enriched CAII variants (Ile, Phe, Leu, and Met at position 93; Ile, Leu, and Met at position 95; and Trp and Val at position 97). Highly selected variants have zinc affinity and catalytic activity nearly equal to that of wild-type CAII, indicating that the aromatic residues are not absolutely essential. However, the zinc dissociation rate constant and catalytic activity of the variants correlate with the volume of the amino acids at positions 93, 95, and 97. In summary, metalloenzyme variants displayed on phage can be selected on the basis of metal affinity; such methods will be useful for optimization of metal ion biosensors.Understanding the structural determinants of metal ion affinity in proteins is an important step in the design of protein metal sites. Such sites may be used to stabilize proteins, to regulate the activity of proteins, or for use in biosensors to quantify trace metal ions (see reviews in Refs. 1-3). Because of its high affinity and specificity for zinc, the His 3 metal polyhedron of carbonic anhydrase (CAII) 1 often has been used as a model for designing metal sites in existing proteins (4 -6) and in de novo proteins such as the minibody (7) and four helical bundle protein (8, 9). While the protein metal ligands of CAII have been incorporated into these proteins with coordination geometry very similar to that observed in CAII, these designed metal sites lack the zinc avidity and catalytic activity of biological zinc sites, suggesting that further protein structural factors in CAII contribute to the metal affinity and reactivity of this enzyme.To probe the relationship between protein structure and metal ion affinity and specificity, many of the conserved structural features near the zinc binding site of CAII have been investigated (review see Ref. 10). The refined x-ray crystal structure of CAII (11) 199 (11). This conserved network of residues that form hydrogen bonds with the His ligands enhances zinc affinity (15-17); the Q92A and E117A substitutions both decrease zinc affinity 5-10-fold and increase the zinc dissociation rate constant. Therefore, the protein structure surrounding the zinc binding site of CAII is finely tuned for high zinc affinity and slow rate constants of metal dissociation.A further structural feature observed in all metalloproteins is that the hydrophilic direct metal ligands are embedded within a larger shell of hydrophobic groups (18). In CAII, residues Phe 93 , Phe 95 , and Trp 97 flank the...

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Section: Discussionmentioning

confidence: 99%

Selection of Carbonic Anhydrase Variants Displayed on Phage

Hunt¹,

Fierke²

1997

Journal of Biological Chemistry

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“…The PCR fragment was ligated into the pCR 2.1 vector (Invitrogen), generating pCIP351. This plasmid was digested with ApaLI and NotI, and the fragment carrying l2-loop was cloned into the corresponding sites of the replicative form of the fd-tet-DOG1 phage (18,19). In the resulting phage, fd-tet-DOG1-L2, the l2-loop was fused with the 5Ј end of the fd-tet-DOG1 gene encoding the G3p protein to generate the l2-loop-g3p gene under the control of the P G3p promoter.…”

Section: Methodsmentioning

confidence: 99%

Evidence of an Intramolecular Interaction between the Two Domains of the BlaR1 Penicillin Receptor during the Signal Transduction

Hanique

Colombo

Goormaghtigh

et al. 2004

Journal of Biological Chemistry

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“…Substitution reaction of disulfide bond of the reagent 1 by a free cysteine residue of proteins is likely to occur more rapidly than the Staudinger ligation reaction between the triarylphosphine moiety of the reagent and azidefunctionality of the proteins. In contrast to this observation of a highly reactive disulfide linker property in reagent 1, a previous study, 19 reported the utility of their reagent compound, which has a disulfide linker between the two functional entities in isolation and selection of their enzymes of interest.…”

Section: Biological Evaluation Of the Biotin-s-s-phosphine (1)mentioning

confidence: 92%

“…3 To ensure the GlcNAz-incorporation of the cellular proteins, the azide-labeled and unlabeled cell lysates were reacted with or without the biotin-conjugated terminal alkyne (19) in combination with copper (I) at room temperature for 20 min (copper-catalyzed 'click' reaction) or Biotin-Phosphine (20) at 37 °C for 1.5 h (Staudinger ligation). Figure 3 shows the reaction results determined by gel electrophoresis follow- ed by Western blot analysis.…”

Section: Biological Evaluation Of the Biotin-s-s-phosphine (1)mentioning

confidence: 99%

Design, Synthesis and Preliminary Biological Evaluation of a Biotin-S-S-Phosphine Reagent

Kang¹,

Kim²

2014

Bulletin of the Korean Chemical Society

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Biotin-S-S-Phosphine was designed and synthesized as a potential tool for a proteomic study of O-GlcNAcmodified proteins. This reagent features a disulfide linker between a triarylphosphine moiety, which allows selective conjugation to azide-containing proteins, and a biotin moiety that can allow easy isolation through its strong affinity toward avidin-coated solid beads. The disulfide linkage within this reagent can allow the easy release of the bound molecules of interest, which is difficult to achieve when a biotin:avidin pair is used alone, by reducing the disulfide bond of the reagent with DTT. Preliminary in vitro biological assays with azidelabeled and unlabeled cell lysates and a pure protein Nup62 showed that the Biotin-S-S-Phosphine reagent is highly reactive toward the free thiol groups of proteins. When a molecular tool with a disulfide linker is applied to the enrichment of the molecules of interest from other species, it is important to block the free-thiols of the sample using exhaustive alkylation prior to the Staudinger ligation reactions to restore the bioorthogonal nature of this reaction.

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Selection of β-Lactamase on Filamentous Bacteriophage by Catalytic Activity

Cited by 126 publications

References 0 publications

Selection of Carbonic Anhydrase Variants Displayed on Phage

Selection of Carbonic Anhydrase Variants Displayed on Phage

Evidence of an Intramolecular Interaction between the Two Domains of the BlaR1 Penicillin Receptor during the Signal Transduction

Design, Synthesis and Preliminary Biological Evaluation of a Biotin-S-S-Phosphine Reagent

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