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General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli : Production of the Bence-Jones protein REIv via fusion to beta-lactamase

General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli : Production of the Bence-Jones protein REIv via fusion to beta-lactamase

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KOLMAR, Harald, Elisa MAY, Frank HENNECKE, Jürgen WIPPLER, Hans-Joachim FRITZ, 1992. General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli : Production of the Bence-Jones protein REIv via fusion to beta-lactamase. In: Journal of Molecular Biology. 228(2), pp. 359-365. ISSN 0022-2836

@article{Kolmar1992Gener-22065, title={General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli : Production of the Bence-Jones protein REIv via fusion to beta-lactamase}, year={1992}, doi={10.1016/0022-2836(92)90826-6}, number={2}, volume={228}, issn={0022-2836}, journal={Journal of Molecular Biology}, pages={359--365}, author={Kolmar, Harald and May, Elisa and Hennecke, Frank and Wippler, Jürgen and Fritz, Hans-Joachim} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/22065"> <dc:contributor>May, Elisa</dc:contributor> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2013-03-20T10:49:04Z</dc:date> <dc:creator>Hennecke, Frank</dc:creator> <dcterms:abstract xml:lang="eng">A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the Vκ domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with β-lactamase as the amino-terminal fusion partner. The β-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.</dcterms:abstract> <dcterms:title>General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli : Production of the Bence-Jones protein REIv via fusion to beta-lactamase</dcterms:title> <dc:creator>Wippler, Jürgen</dc:creator> <dc:contributor>Wippler, Jürgen</dc:contributor> <dc:rights>deposit-license</dc:rights> <dc:contributor>Hennecke, Frank</dc:contributor> <dc:language>eng</dc:language> <dcterms:issued>1992</dcterms:issued> <dcterms:rights rdf:resource="http://nbn-resolving.org/urn:nbn:de:bsz:352-20140905103605204-4002607-1"/> <dc:creator>Fritz, Hans-Joachim</dc:creator> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/22065"/> <dc:contributor>Fritz, Hans-Joachim</dc:contributor> <dc:creator>May, Elisa</dc:creator> <dc:creator>Kolmar, Harald</dc:creator> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2013-03-20T10:49:04Z</dcterms:available> <dcterms:bibliographicCitation>Journal of molecular biology ; 228 (1992), 2. - S. 359-365</dcterms:bibliographicCitation> <dc:contributor>Kolmar, Harald</dc:contributor> </rdf:Description> </rdf:RDF>

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