Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats

Pershad, Kritika; Sullivan, Mark A.; Kay, Brian K.

doi:10.1016/j.ab.2011.02.006

Cited by 12 publications

(14 citation statements)

References 36 publications

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“…The coding sequence of FN3 monobody was subcloned into a new phagemid vector [37], fusing it with the truncated gene III of M13 bacteriophage, which allows monovalent display of the monobody protein on the phage surface. Diversifying the 5 residues of both the BC and FG loops of FN3 scaffold (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, the FN3 coding region was subcloned into a drop-out phagemid vector [37], which encodes a truncated form of gene III of the M13 bacteriophage and the DsbA signal sequence [38]. The recombinant phagemid construct was transformed into the bacterial strain CJ236 (New England BioLabs, Ipswich, MA), an Escherichia coli host used for producing uracil-containing single-stranded DNA (ssDNA).…”

Section: Methodsmentioning

confidence: 99%

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Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

2012

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Fyn is a non-receptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases (SFKs). Fyn plays an important role in inflammatory processes and neuronal functions. To generate a synthetic affinity reagent that can be used to probe Fyn, a phage-display library of fibronectin type III (FN3) monobodies was affinity selected with the SH3 domain of Fyn and three binders were isolated. One of the three binders, G9, is specific in binding to the SH3 domain of Fyn, but not to the other members of the Src family (i.e., Blk, Fgr, Hck, Lck, Lyn, Src, Yes), even though they share 51-81% amino acid identity. The other two bind principally to the Fyn SH3 domain, with some cross-reactivity to the Yes SH3 domain. The G9 binder has a dissociation constant of 166 ± 6 nM, as measured by isothermal titration calorimetry, and binds only to the Fyn SH3 domain out of 150 human SH3 domains examined in an array. Interestingly, although the G9 monobody lacks proline in its randomized BC and FG loops, it binds at the same site on the SH3 domain as proline-rich ligands, as revealed by competition assays. The G9 monobody, identified in this study, may be used as a highly selective probe for detecting and purifying cellular Fyn kinase.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

2012

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“…The sequence of the 10 th subunit of human fibronectin type III repeat (FN3) was amplified by PCR from a plasmid [21], and subcloned into the pAP-III 6 vector [22, 23]. In this phagemid vector, the Flag (DYKDDDDK) epitope is fused at the N-terminus of the FN3 coding region, thereby allowing convenient detection of the displayed FN3 domain with an anti-Flag antibody (Sigma-Aldrich; St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

Huang

Fang

Kay

2012

Methods

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Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded 2- to 7-fold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound 2- to 4-fold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents.

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“…The 3C-3S coding sequence was amplified by polymerase chain reaction (PCR), using primers FHA1-NcoI-Fw and FHA1-NotI-Rv and the AccuPrime™ Pfx DNA polymerase (Invitrogen), for creating flanking Nco I/ Not I sites for subcloning into the phagemid vector (pKP600) in-frame with the gene III coding sequence. The phagemid vector 42 used is a modified version of the pKP300 vector except that it has a DsbA signal sequence and lacks the alkaline phosphatase coding sequence. All the primers were ordered from Integrated DNA Technologies and their sequences are listed in Table S4.…”

Section: Methodsmentioning

confidence: 99%

Directed Evolution of the Forkhead-Associated Domain to Generate Anti-Phosphospecific Reagents by Phage Display

Pershad

Wypisniak

Kay

2012

Journal of Molecular Biology

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While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display.

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Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats

Cited by 12 publications

References 36 publications

Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

Directed Evolution of the Forkhead-Associated Domain to Generate Anti-Phosphospecific Reagents by Phage Display

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