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Titolo:
Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli
Autore:
Richter, W; Hermsdorf, T; Lilie, H; Egerland, U; Rudolph, R; Kronbach, T; Dettmer, D;
Indirizzi:
Univ Leipzig, Fac Med, Inst Biochem, D-04103 Leipzig, Germany Univ Leipzig Leipzig Germany D-04103 t Biochem, D-04103 Leipzig, Germany Univ Halle Wittenberg, Inst Biotechnol, D-06120 Halle, Germany Univ Halle Wittenberg Halle Germany D-06120 hnol, D-06120 Halle, Germany ASTA Med, Biochem Dresden, D-01445 Radebeul, Germany ASTA Med Radebeul Germany D-01445 hem Dresden, D-01445 Radebeul, Germany
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 3, volume: 19, anno: 2000,
pagine: 375 - 383
SICI:
1046-5928(200008)19:3<375:RPACOH>2.0.ZU;2-7
Fonte:
ISI
Lingua:
ENG
Soggetto:
CAMP-SPECIFIC PHOSPHODIESTERASE; ROLIPRAM; BINDING; ACTIVATION; PROTEINS; CLONING; DRUGS; CELLS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
23
Recensione:
Indirizzi per estratti:
Indirizzo: Dettmer, D Univ Leipzig, Fac Med, Inst Biochem, Liebigstr 16, D-04103 Leipzig, Germany Univ Leipzig Liebigstr 16 Leipzig Germany D-04103 zig, Germany
Citazione:
W. Richter et al., "Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli", PROT EX PUR, 19(3), 2000, pp. 375-383

Abstract

A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR, Several PDE4A constructs containing the catalytic region and differing in their degree: of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptidesaccumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results, Thebest results were obtained with the Nand C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and wasfound to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructsobtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates, (C) 2000 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 23/01/21 alle ore 10:23:54