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Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO), Bioprocess Technology.
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
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2005 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 69, no 1, 92-98 p.Article in journal (Refereed) Published
Abstract [en]

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

Place, publisher, year, edition, pages
2005. Vol. 69, no 1, 92-98 p.
Keyword [en]
antibacterial peptides, expression, membrane, protein, growth
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-5225DOI: 10.1007/s00253-005-1958-6ISI: 000233502300014Scopus ID: 2-s2.0-28344454084OAI: oai:DiVA.org:kth-5225DiVA: diva2:8058
Note
QC 20100830 Uppdaterat från manuskript till artikel (20100830)Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Physiological effects of conditioned medium and passage number on Spodoptera frugiperda Sf9 serum free cultures
Open this publication in new window or tab >>Physiological effects of conditioned medium and passage number on Spodoptera frugiperda Sf9 serum free cultures
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system.

CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction.

Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. 43 p.
Keyword
Biotechnology, Spodoptera frugiperda Sf9, insect cell cultures, conditioned medium, cell cycle phase dynamics, proteolytic activity, antibacterial activity, recombinant protein production, Bioteknik
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-247 (URN)91-7178-074-2 (ISBN)
Presentation
2005-06-01, FA32, AlbaNova, Roslagstullsbacken 21B, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101222Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2012-03-21Bibliographically approved
2. Physiology and productivity of serum-free Spodoptera frugiperda Sf9 insect cell cultures
Open this publication in new window or tab >>Physiology and productivity of serum-free Spodoptera frugiperda Sf9 insect cell cultures
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The objective of this study was to investigate the mechanisms and factors controlling growth and proliferation in serum-free Spodoptera frugiperda Sf9 cultures as well as the implications of these factors for protein production in the baculovirus expression system.

The physiology of recently thawed, low passage (Lp) Sf9 cultures, were compared to high passage (Hp) cultures at p>100. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µN,max, from 0.03/h to 0.04/h. Conditioned medium (CM), 10 kDa CM filtrate and 10 kDa CM concentrate promoted proliferation of Lp Sf9 cells, but had no effect after the switch. Sf9 cell cycle dynamics were characterized by an initial G2/M arrest, which synchronized the cells and this feature was more pronounced for Hp than for Lp cells. CM addition decreased the initial arrest for Lp cultures, but did not affect Hp cells. Late in the culture, a final G2/M accumulation occurred. An octaploid population emerged during G2/M arrests. Further, a 49 kDa proform and a 39 kDa active form of Sf9 cathepsin L was identified from gelatine zymography of Sf9 CM, on basis of inhibitor profile and substrate range. Removal of procathepsin L during the course of an Sf9 culture had a negative effect on Sf9 proliferation. Procathepsin L was also identified in Trichoplusia ni High five CM. High five CM promoted growth of Sf9 cells, but when procathepsin L was removed no effect was observed. It is suggested that these observations are due to an autocrine system controlling proliferation. One Sf9 mitogen might be a <10 kDa peptide, while the effect of 10 kDa CM concentrate may originate from procathepsin L. A hypothesis is therefore that procathepsin L acts as a mitogen in Sf9 cultures, perhaps in concert with the <10 kDa peptide.

The volumetric product yield (P) in baculovirus infected Sf9 cells increased linearly up to 68-75 h of culture. Beyond this point almost no product was detected. Medium renewal at infection prolonged the productivity phase until 117 h, but generated only a 10% increase in P. The specific product formation rate (YP/N) was highest at µN,max. YP/N of Lp cells decreased by 30-50% when 20% CM or 10 kDa CM filtrate was added, whereas addition of CM to cells having passed the switch on growth kinetics did not affect productivity. Further, Hp cells exhibited a two-fold higher YP/N than Lp cells, when infected during the initial 48 h of culture. This coincided with a high degree of synchronization. Yeastolate limitation was used to achieve artificial synchronization of an Lp culture, and YP/N could thereby be maintained high during a prolonged time, resulting in a 69% increased P. This suggests that a decreasing degree of synchronization during the course of a culture partly explains the cell-density dependent drop in productivity in Sf9 cells.

Finally, ~10 kDa gel filtration fractions from Sf9 and High five CM were found to be bactericidal. Exposure of a Bacillus megaterium culture for eight min to an Sf9 CM fraction killed 99% of the population, and 60 min exposure killed 35% of an Escherichia coli population. In both cases cell lysis was observed. B. megaterium incubated in an High five CM fraction lost 97% viability in 40 min. The effect of the High five CM fraction most probably originated from a lysozyme precursor protein, whereas the Sf9 executor remains unknown.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 53 p.
Keyword
Animalcellteknologi
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4033 (URN)91-7178-383-0 (ISBN)
Public defence
2006-06-15, Sal FB52, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100908Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2010-09-08Bibliographically approved

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