title: Proteolytic activity in infected and noninfected insect cells: degradation of HIV-1 Pr55gag particles.
abstract: In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It was observed that infection caused not only a reduction in the concentration of proteases by decreasing their synthesis but also an inhibition of the intracellular proteolytic activity by increasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was observed at 72 hpi except when the cells were infected in the late exponential growth phase or with very low MOI, yielding a nonsynchronous infection. The proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supernatant should be stored at low temperature or quickly purified, since the degradation after 24 h is only 3% at 4 degrees C while at 27 degrees C this value rises to 23%. There is a complex relationship between MOI, TOI, proteolytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the desired product quality, and operational issues like process time and capacity, requiring proper integration between bioreaction and downstream processing.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, IBET/ITQB, Apartado 12, P-2780 Oeiras, Portugal.
authors.forename: P E
authors.initials: PE
authors.lastname: Cruz
authors.forename: P C
authors.initials: PC
authors.lastname: Martins
authors.forename: P M
authors.initials: PM
authors.lastname: Alves
authors.forename: C C
authors.initials: CC
authors.lastname: Peixoto
authors.forename: H
authors.initials: H
authors.lastname: Santos
authors.forename: J L
authors.initials: JL
authors.lastname: Moreira
authors.forename: M J
authors.initials: MJ
authors.lastname: Carrondo
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis; D000418:Albumins / Q000378:metabolism; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002478:Cells, Cultured; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015683:Gene Products, gag / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism / Q000472:pathogenicity*; D009682:Magnetic Resonance Spectroscopy; D011498:Protein Precursors / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology*; D013997:Time Factors; D014162:Transfection
chemical_list: D000418:Albumins; D002364:Caseins; D005456:Fluorescent Dyes; D015683:Gene Products, gag; D011498:Protein Precursors; C031460:azoalbumin; C026018:azocasein; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D016650:Fluorescein-5-isothiocyanate