Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system

1992 ◽  
Vol 8 (5) ◽  
pp. 391-396 ◽  
Author(s):  
T. J. Wickham ◽  
T. Davis ◽  
R. R. Granados ◽  
M. L. Shuler ◽  
H. A. Wood
Keyword(s):  
Cell Lines ◽  
Recombinant Proteins ◽  
Insect Cell ◽  
Infectious Virus ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Baculovirus Expression ◽  
Insect Cell Lines

Expression and characterization of recombinant human α-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system

2001 ◽  
Vol 353 (3) ◽  
pp. 719-725 ◽  
Author(s):  
Vanessa A. MORAIS ◽  
Jacinta SERPA ◽  
Angelina S. PALMA ◽  
Teresa COSTA ◽  
Luís MARANGA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Trichoplusia Ni ◽  
Secretory Pathway ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Exchange Chromatography ◽  
Sf9 Cells ◽  
Type I ◽  
Baculovirus Expression ◽  
Insect Cell Lines

The human α-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodopterafrugiperda (Sf9) and Trichoplusiani (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4unit/l (1mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were kcat = 0.4s-1 and Km = 0.87mM for the SFT3 secreted by Tn cells, and kcat = 0.09s-1 and Km = 0.76mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII.

Production of therapeutic proteins with baculovirus expression system in insect cell

2008 ◽  
Vol 38 ◽  
pp. S71-S78 ◽  
Author(s):  
Mi-Hyun AHN ◽  
Mira SONG ◽  
Eun-Yi OH ◽  
Arshad JAMAL ◽  
HyunSoon KIM ◽  
...  
Keyword(s):  
Insect Cell ◽  
Expression System ◽  
Therapeutic Proteins ◽  
Baculovirus Expression System ◽  
Baculovirus Expression

High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line

1996 ◽  
Vol 17 (2) ◽  
pp. 165-174 ◽  
Author(s):  
F Grennan Jones ◽  
A Wolstenholme ◽  
S Fowler ◽  
S Smith ◽  
K Ziemnicka ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Insect Cell ◽  
Culture Medium ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
Insect Cell Line ◽  
Thyroid Peroxidase ◽  
High Five Cells ◽  
Insect Cell Lines

ABSTRACT Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotin-labelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r=0·96, P<0·001, n=50 and r=0·99, P<0·001, n=80 respectively). In addition, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor δ-aminolevulinic acid was included in the culture medium. Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO—TPO autoantibody complexes.

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