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Titolo:
HIGH-YIELD EXPRESSION AND PURIFICATION OF HUMAN ENDOTHELIN-1
Autore:
FASSINA G; MERLI S; GERMANI S; CILIBERTO G; CASSANI G;
Indirizzi:
TECNOGEN SCPA,PROT ENGN UNIT,PARCO SCI I-81015 PIANA MONTE VERNA ITALY TECNOGEN SCPA,MOLEC BIOL UNIT PIANA MONTE VERNA ITALY UNIV NAPLES,DIPARTIMENTO BIOCHIM & BIOTECNOL MED NAPLES ITALY
Titolo Testata:
Protein expression and purification
fascicolo: 6, volume: 5, anno: 1994,
pagine: 559 - 568
SICI:
1046-5928(1994)5:6<559:HEAPOH>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
HYDROPATHICALLY COMPLEMENTARY; RECOGNITION PROPERTIES; ANTISENSE PEPTIDES; CONVERTING ENZYME; ESCHERICHIA-COLI; BIG ENDOTHELIN; PRECURSOR; PROTEIN; INHIBITORS; SEQUENCES;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
28
Recensione:
Indirizzi per estratti:
Citazione:
G. Fassina et al., "HIGH-YIELD EXPRESSION AND PURIFICATION OF HUMAN ENDOTHELIN-1", Protein expression and purification, 5(6), 1994, pp. 559-568

Abstract

A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recogni- tion site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 mu g/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 mu g of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%. (C) 1984 Academic Press, Inc,

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/04/20 alle ore 21:41:30