The rational construction of an antibody against cystatin: lessons from the crystal structure of an artificial Fab Fragment

Schiweck, Wolfram; Skerra, Arne

doi:10.1006/jmbi.1997.1006

Cited by 18 publications

(11 citation statements)

References 48 publications

(63 reference statements)

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“…Altogether 19 side chains were replaced within the CDRs for this purpose, whereby a five-residue insertion was introduced into CDR-L1. The X-ray crystallographic analysis of the resulting artifical F ab fragment 'M41' revealed indeed close similarity with the modelled structure (Schiweck and Skerra, 1997). However, some local deviations were also seen, explaining the lack of affinity for the new antigen at this stage.…”

Section: Introduction: What Is a Molecular 'Scaffold'?mentioning

confidence: 53%

Engineered protein scaffolds for molecular recognition

Skerra

2000

J. Mol. Recognit.

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The use of so-called protein scaffolds has recently attracted considerable attention in biochemistry in the context of generating novel types of ligand receptors for various applications in research and medicine. This development started with the notion that immunoglobulins owe their function to the composition of a conserved framework region and a spatially well-defined antigen-binding site made of peptide segments that are hypervariable both in sequence and in conformation. After the application of antibody engineering methods along with library techniques had resulted in first successes in the selection of functional antibody fragments, several laboratories began to exploit other types of protein architectures for the construction of practically useful binding proteins. Properties like small size of the receptor protein, stability and ease of production were the focus of this work. Hence, among others, single domains of antibodies or of the immunoglobulin superfamily, protease inhibitors, helix-bundle proteins, disulphide-knotted peptides and lipocalins were investigated. Recently, the scaffold concept has even been adopted for the construction of enzymes. However, it appears that not all kinds of polypeptide fold which may appear attractive for the engineering of loop regions at a first glance will indeed permit the construction of independent ligand-binding sites with high affinities and specificities. This review will therefore concentrate on the critical description of the structural properties of experimentally tested protein scaffolds and of the novel functions that have been achieved on their basis, rather than on the methodology of how to best select a particular mutant with a certain activity. An overview will be provided about the current approaches, and some emerging trends will be identified. (c) 2000 John Wiley & Sons, Ltd. Abbreviations used: ABD albumin-binding domain of protein G APPI Alzheimer's amyloid beta-protein precursor inhibitor BBP bilin-binding protein BPTI bovine (or basic) pancreatic trypsin inhibitor BSA bovine serum albumin CBD cellulose-binding domain of cellobiohydrolase I CD circular dichroism Cdk2 human cyclin-dependent kinase 2 CDR complementarity-determining region CTLA-4 human cytotoxic T-lymphocyte associated protein-4 FN3 fibronectin type III domain GSH glutathione GST glutathione S-transferase hIL-6 human interleukin-6 HSA human serum albumin IC(50) half-maximal inhibitory concentration Ig immunoglobulin IMAC immobilized metal affinity chromatography K(D) equilibrium constant of dissociation K(i) equilibrium dissociation constant of enzyme inhibitor LACI-D1 human lipoprotein-associated coagulation inhibitor pIII gene III minor coat protein from filamentous bacteriophage f1 PCR polymerase-chain reaction PDB Protein Data Bank PSTI human pancreatic secretory trypsin inhibitor RBP retinol-binding protein SPR surface plasmon resonance TrxA E. coli thioredoxin

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Section: Introduction: What Is a Molecular 'Scaffold'?mentioning

confidence: 53%

Engineered protein scaffolds for molecular recognition

Skerra

2000

J. Mol. Recognit.

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“…3B). Specific elution of the bound protein was achieved under gentle buffer conditions by the application of desthiobiotin in this case (Voss & Skerra, 1997). The protein preparations from either chromatography were indistinguishable in terms of yield and quality.…”

Section: Bacterial Production and Purification Of The Cystatin-gstfusionmentioning

confidence: 99%

“…The N-terminal peptide segment as well as two internal loops are involved in the complex formation with its cognate protease papain. The same region was chosen as an epitope when cystatin was employed in a protein design study with an antibody (Schiweck & Skerra, 1997). To make GST amenable as a dimerization module for cystatin it had thus to be tested whether GST is still a functional enzyme when another protein gets fused to its N-terminal end.…”

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confidence: 99%

Glutathione S‐transferase can be used as a C‐terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli

Tudyka

Skerra

1997

Protein Science

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Glutathione S-transferase (GST) from Schistosoma juponicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C-terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and l-chloro-2,4-dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity.

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“…After generation of cDNA, the hypervariable sequences of heavy and light chains were amplified as described before [21] and verified by sequencing. The obtained variable (V) domains from the heavy (V H ) and light (V L ) chains were cloned on a pENTRY-IBA50 StarGate vector allowing the combination with sequences coding for the constant domains of human subclass IgG1/κ [22] in a subsequent recombination step. The heavy chains were carboxy-terminally fused with a OneSTrEPtag affinity tag (IBA).…”

Section: Methodsmentioning

confidence: 99%

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

Stemberger

Dreher

Tschulik³

et al. 2012

PLoS ONE

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A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

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The rational construction of an antibody against cystatin: lessons from the crystal structure of an artificial Fab Fragment

Cited by 18 publications

References 48 publications

Engineered protein scaffolds for molecular recognition

Engineered protein scaffolds for molecular recognition

Glutathione S‐transferase can be used as a C‐terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

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