(Research Papers) Accelerated Screening of Phage-Display Output with Alkaline Phosphatase Fusions

Han, Zhibin; Karatan, Ece; Scholle, Michael D.; McCafferty, John; Kay, Brian K.

doi:10.2174/138620704772884823

Cited by 22 publications

(18 citation statements)

References 35 publications

(51 reference statements)

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“…Alkaline phosphatase fusion proteins of affinity clamps were made by cloning the alkaline phosphatase gene (a gift of Brian Kay, University of Illinois, Chicago, IL) (40) to the 3Ј of the affinity clamp gene in the phage display vector described above in such a way that the alkaline phosphatase replaces the phage pIII protein. The fusion proteins were expressed in XL1-Blue cells, and the periplasmic fraction containing AP-affinity clamp fusion proteins was used for detection.…”

Section: Construction Of Phage Display Vectors and Combinatorial Librmentioning

confidence: 99%

Design of protein function leaps by directed domain interface evolution

Huang

Koide

Makabe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

107

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Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting ''affinity clamp'' had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.affinity reagent ͉ epitope ͉ molecular evolution ͉ protein design ͉ PDZ domain D irected evolution-based protein engineering creates new protein functions by exploiting processes that occur during natural evolution of proteins. Protein evolution progresses via point mutations, duplication, and recombination of genes under selective pressure. The processes of gene duplication and subsequent sequence divergence (1) have been successfully recapitulated in directed evolution and computational protein design (2, 3) where preexisting active sites within natural protein scaffolds are altered to produce new functions (Fig.

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Section: Construction Of Phage Display Vectors and Combinatorial Librmentioning

confidence: 99%

Design of protein function leaps by directed domain interface evolution

Huang

Koide

Makabe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

107

View full text Add to dashboard Cite

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“…As the purified scFv was poorly expressed, two recombinant forms of greater stability were created: full-length IgG and an scFv-AP fusion protein (61,(72)(73)(74)(75). These are both dimeric and were found to be stable for months at 4°C.…”

Section: Synthesis Of the Peptide Antigen-two Peptide Sequencesmentioning

confidence: 99%

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Kehoe

Velappan

Walbolt

et al. 2006

Molecular & Cellular Proteomics

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Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Much protein activity is regulated by post-translational modifications (PTMs), 1 over 200 of which have been described (1), with changes in enzymatic activity, protein interaction partners, subcellular localization, and targeted degradation being some of the most important regulated activities. A number of different approaches have been used to study PTMs, most of which rely on either specific isolation of the protein of interest and identification of the PTMs that affect that particular protein (2) or isolation of all proteins containing a specific PTM of interest and subsequent identification of the isolated proteins. There are two basic methods to isolate or identify all proteins containing a specific PTM: chemical derivatization or specific affinity reagents. The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5-8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17). Although antibodies are by far the most common specific affinity reagents, there are very few that recognize PTMs independently of the proteins to which they are attached. Nitrosylated tyrosine and phosphorylated tyrosine are two exceptions for which antibodies have been used in proteomics studies (13-17), and antibodies against phosphoserine/threonine have also been identified (18), indicating that the utility of such reagents...

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“…1). This technology was further developed and improved to display large proteins such as enzymes and antibodies (Fernandez-Gacio et al, 2003;Han et al, 2004). The connection between genotype and phenotype enables large libraries of peptides or proteins to be screened in a relative fast and economic way.…”

Section: Principle Of Phage Displaymentioning

confidence: 99%

“…It can be used for finding new ligands, such as enzyme inhibitors, receptor agonists and antagonists, to target proteins (Hariri et al, 2008;Pasqualini et al, 1995;Perea et al, 2004;Ruoslahti, 1996;Uchino et al, 2005). Invention of antibody phage display revolutionized the drug discovery (Han et al, 2004). Millions of different single chain antibodies on phages are used for isolating highly specific therapeutic antibody leads.…”

Section: Applications Of Phage Display 421 General Applicationsmentioning

confidence: 99%