A Single-Step Purification of Biologically Active Recombinant Human Interleukin-5 from a Baculovirus Expression System

1995 ◽  
Vol 6 (1) ◽  
pp. 63-71 ◽  
Author(s):  
P.M. Brown ◽  
M.P. Scheid ◽  
G.P. Oneil ◽  
P.C. Tagari ◽  
D.W. Nicholson
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Single Step ◽  
Biologically Active ◽  
Interleukin 5 ◽  
Baculovirus Expression ◽  
Single Step Purification

Expression of a biologically active ovine trophoblastic interferon using a baculovirus expression system

1991 ◽  
Vol 181 (1) ◽  
pp. 443-448 ◽  
Author(s):  
Martine Cerutti ◽  
Dominique Hue ◽  
Madia Charlier ◽  
René L'Haridon ◽  
Jean-Claude Pernollet ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Biologically Active ◽  
Baculovirus Expression

Purification and characterisation of recombinant murine interleukin-5 glycoprotein, from a Baculovirus expression system

1993 ◽  
Vol 21 (4) ◽  
pp. 332S-332S ◽  
Author(s):  
DIANE L. MITCHELL ◽  
MOIRA A. YOUNG ◽  
CLAIRE ENTWISLE ◽  
ANNETTE N. DAVIES ◽  
RICHARD M. COOK ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Interleukin 5 ◽  
Baculovirus Expression

scholarly journals Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system

1996 ◽  
Vol 314 (2) ◽  
pp. 511-519 ◽  
Author(s):  
Sheryl L. MEYER ◽  
Donna BOZYCZKO-COYNE ◽  
Satish K. MALLYA ◽  
Chrysanthe M. SPAIS ◽  
Ron BIHOVSKY ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Cysteine Proteases ◽  
Catalytic Subunit ◽  
Biologically Active ◽  
Regulatory Subunit ◽  
Calcium Dependent ◽  
Autocatalytic Cleavage ◽  
Cell Extracts ◽  
Baculovirus Expression

Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The ~80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the ~30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of ~80 kDa subunit accumulated at steady state was greatly increased by co-expression of the ~30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.

scholarly journals The Pfs230 N-terminal fragment, Pfs230D1+: expression and characterization of a potential malaria transmission-blocking vaccine candidate

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Shwu-Maan Lee ◽  
Yimin Wu ◽  
John M. Hickey ◽  
Kazutoyo Miura ◽  
Neal Whitaker ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Glycosylation Site ◽  
Biologically Active ◽  
Size Exclusion ◽  
Intact Protein ◽  
Transmission Blocking ◽  
Baculovirus Expression ◽  
Sds Page ◽  
Transmission Blocking Vaccine

Abstract Background Control and elimination of malaria can be accelerated by transmission-blocking interventions such as vaccines. A surface antigen of Plasmodium falciparum gametocytes, Pfs230, is a leading vaccine target antigen, and has recently progressed to experimental clinical trials. To support vaccine product development, an N-terminal Pfs230 antigen was designed to increase yield, as well as to improve antigen quality, integrity, and homogeneity. Methods A scalable baculovirus expression system was used to express the Pfs230D1+ construct (aa 552–731), which was subsequently purified and analysed. Pfs230D1+ was designed to avoid glycosylation and protease digestion, thereby potentially increasing homogeneity and stability. The resulting Pfs230D1+ protein was compared to a previous iteration of the Pfs230 N-terminal domain, Pfs230C1 (aa 443–731), through physiochemical characterization and in vivo analysis. The induction of functional antibody responses was confirmed via the standard membrane feeding assay (SMFA). Results Pfs230D1+ was produced and purified to an overall yield of 23 mg/L culture supernatant, a twofold yield increase over Pfs230C1. The Pfs230D1+ protein migrated as a single band via SDS-PAGE and was detected by anti-Pfs230C1 monoclonal antibodies. Evaluation by SDS-PAGE, chromatography (size-exclusion and reversed phase) and capillary isoelectric focusing demonstrated the molecule had improved homogeneity in terms of size, conformation, and charge. Intact mass spectrometry confirmed its molecular weight and that it was free of glycosylation, a key difference to the prior Pfs230C1 protein. The correct formation of the two intramolecular disulfide bonds was initially inferred by binding of a conformation specific monoclonal antibody and directly confirmed by LC/MS and peptide mapping. When injected into mice the Pfs230D1+ protein elicited antibodies that demonstrated transmission-reducing activity, via SMFA, comparable to Pfs230C1. Conclusion By elimination of an O-glycosylation site, a potential N-glycosylation site, and two proteolytic cleavage sites, an improved N-terminal Pfs230 fragment was produced, termed D1+, which is non-glycosylated, homogeneous, and biologically active. An intact protein at higher yield than that previously observed for the Pfs230C1 fragment was achieved. The results indicate that Pfs230D1+ protein produced in the baculovirus expression system is an attractive antigen for transmission-blocking vaccine development.

High yield of biologically active recombinant human amelogenin using the baculovirus expression system

2006 ◽  
Vol 45 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Angela L. Taylor ◽  
Amir Haze-Filderman ◽  
Anat Blumenfeld ◽  
Boaz Shay ◽  
Leah Dafni ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Biologically Active ◽  
High Yield ◽  
Baculovirus Expression

scholarly journals Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

1992 ◽  
Vol 267 (19) ◽  
pp. 13123-13126 ◽  
Author(s):  
S.G. Graber ◽  
R.A. Figler ◽  
V.K. Kalman-Maltese ◽  
J.D. Robishaw ◽  
J.C. Garrison
Keyword(s):  
G Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Subunit Composition ◽  
Baculovirus Expression

scholarly journals Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Partial Purification ◽  
Baculovirus Expression ◽  
Gamma Subunits ◽  
Infected Insect ◽  
Beta 2

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.

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