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Titolo:
Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors
Autore:
Gotoh, T; Miyazaki, Y; Sato, W; Kikuchi, KI; Bentley, WE;
Indirizzi:
Akita Univ, Dept Mat Proc Engn & Appl Chem Environm, Akita 0108502, Japan Akita Univ Akita Japan 0108502 Appl Chem Environm, Akita 0108502, Japan Univ Maryland, Ctr Agr Biotechnol, College Pk, MD 20742 USA Univ MarylandCollege Pk MD USA 20742 iotechnol, College Pk, MD 20742 USA Univ Maryland, Dept Chem Engn, College Pk, MD 20742 USA Univ Maryland College Pk MD USA 20742 Chem Engn, College Pk, MD 20742 USA
Titolo Testata:
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
fascicolo: 3, volume: 92, anno: 2001,
pagine: 248 - 255
SICI:
1389-1723(200109)92:3<248:PAARPP>2.0.ZU;2-2
Fonte:
ISI
Lingua:
ENG
Soggetto:
GREEN FLUORESCENT PROTEIN; BACULOVIRUS EXPRESSION SYSTEMS; GENE; DEGRADATION; PROTECTION; PARTICLES; MARKER;
Keywords:
baculovirus; insect cell; Sf-9; protease; protease inhibitor;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
28
Recensione:
Indirizzi per estratti:
Indirizzo: Gotoh, T Akita Univ, Dept Mat Proc Engn & Appl Chem Environm, Akita 0108502, Japan Akita Univ Akita Japan 0108502 m Environm, Akita 0108502, Japan
Citazione:
T. Gotoh et al., "Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors", J BIOSCI BI, 92(3), 2001, pp. 248-255

Abstract

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 andpepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatinA which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 06/04/20 alle ore 01:54:38