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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
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- 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