pSKAP/S: An Expression Vector for the Production of Single-Chain Fv Alkaline Phosphatase Fusion Proteins

Griep, R.A.; Twisk, C. van; Kerschbaumer, Randolf J.; Harper, K.; Torrance, L.; Himmler, Gottfried; Wolf, J.M. van der; Schots, A.

doi:10.1006/prep.1999.1041

Cited by 46 publications

(24 citation statements)

References 16 publications

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“…In order to overcome this difficulty, (especially during the selection of scFv (single-chain fragment variable) from phage display libraries), scFv (single-chain fragment variable) in a phage format can be used in ELISA assay, which means that the scFv (single-chain fragment variable) is remain attached to the coat protein of filamentous phage. By applying this step, it may improve the protein folding as the phage may mimic missing constant domains of the original antibody [148]. …”

Section: Application Of Single-chain Fragment Variable (Scfv) and mentioning

confidence: 99%

scFv Antibody: Principles and Clinical Application

Ahmad

Yeap

Ali

et al. 2012

Clinical and Developmental Immunology

593

451

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To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

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Section: Application Of Single-chain Fragment Variable (Scfv) and mentioning

confidence: 99%

scFv Antibody: Principles and Clinical Application

Ahmad

Yeap

Ali

et al. 2012

Clinical and Developmental Immunology

593

451

View full text Add to dashboard Cite

show abstract

“…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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“…Theoretically, it is possible to prepare antibodies fused with proteins other than fluorescent protein. For example, fusion of primary antibodies with peroxidase or alkaline phosphatase [41] will provide useful tools for enzyme-linked immunological assays, suggesting further applications to diagnostic purposes.…”

Section: Discussionmentioning

confidence: 99%

B Cell-Based Seamless Engineering of Antibody Fc Domains

et al. 2016

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Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.

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pSKAP/S: An Expression Vector for the Production of Single-Chain Fv Alkaline Phosphatase Fusion Proteins

Cited by 46 publications

References 16 publications

scFv Antibody: Principles and Clinical Application

scFv Antibody: Principles and Clinical Application

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

B Cell-Based Seamless Engineering of Antibody Fc Domains

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