Effects of Co-Expressing Chaperone BiP on Functional Antibody Production in the Baculovirus System

Hsu, Tsu‐An; Eiden, Joseph; Bourgarel, P; Meo, Tommaso; Betenbaugh, Michael J

doi:10.1006/prep.1994.1082

Cited by 60 publications

(50 citation statements)

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“…In Aspergillus awamori, elevated BiP levels did not show any significant effect on the secretion efficiency of different cutinase variants (van Gemeren et al 1998). Similar results were observed in the case of secretion of heterologous proteins by mammalian cells (Dorner et al 1992;Hsu et al 1994).…”

Section: Discussionsupporting

confidence: 85%

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

Heide

Hollenberg

Klei

et al. 2002

Applied Microbiology and Biotechnology

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Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 11-05-2018Abstract We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomyces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved -HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (P MOX ), on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low P MOX induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains.

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Section: Discussionsupporting

confidence: 85%

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

Heide

Hollenberg

Klei

et al. 2002

Applied Microbiology and Biotechnology

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“…Alkaline phosphataseconjugated goat anti-rabbit antibody was from Bio-Rad. Recombinant baculoviruses expressing ER p57 , CRT, CNX, PDI, or BiP were prepared as described previously (32)(33)(34)(35)(36). Antibodies against each of the chaperones were from StressGen (Victoria, Canada).…”

Section: Methodsmentioning

confidence: 99%

Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

Zhang

Tate

et al. 2003

Journal of Biological Chemistry

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Lipoprotein lipase (LPL) is a non-covalent, homodimeric, N-glycosylated enzyme important for metabolism of blood lipids. LPL is regulated by yet unknown post-translational events affecting the levels of active dimers. On co-expression of LPL with human molecular chaperones, we found that calreticulin had the most pronounced effects on LPL activity, but calnexin was also effective. Calreticulin caused a 9-fold increase in active LPL, amounting to about 50% of the expressed LPL protein. The total expression of LPL protein was increased less than 20%, and the secretion rates for active and inactive LPL were not significantly changed by the chaperone. Thus, the main effect was an increased specific activity of LPL both in cells and media. Chromatography on heparin-Sepharose and sucrose density gradient centrifugation demonstrated that most of the inactive LPL was monomeric and that calreticulin promoted formation of active dimers. Higher oligomers of inactive LPL were present in cell extracts, but only monomers and dimers were secreted to the medium. Interaction between LPL and calreticulin was demonstrated, and the effect of the chaperone was prevented by castanospermine, an inhibitor of N-glycan glucose trimming. Our data indicate an important role of endoplasmic reticulum-based chaperones for the folding/ dimerization of LPL. Lipoprotein lipase (LPL)1 hydrolyzes triglycerides in the circulating plasma lipoproteins, such as chylomicrons and very low density lipoproteins (1-3). The enzyme is produced and secreted mainly by adipocytes and muscle cells, but it is also produced in other cells such as macrophages and certain neurons. LPL is transported by yet unclear mechanisms to the vascular endothelium, where it is attached to cell surfaces through interaction with heparan sulfate proteoglycans (HSPG). The products of lipolysis are taken up for storage or oxidation in adjacent tissues. In addition, LPL can mediate internalization of intact lipoproteins by bridging them to cell surface receptors like the LDL receptor-like protein or HSPG.LPL, along with pancreatic lipase, hepatic lipase (HL), and endothelial lipase, belongs to the gene family of mammalian triglyceride lipases (2, 4). The three-dimensional structure of LPL has not yet been determined. Based on homology with pancreatic lipase, it has been proposed that LPL has two structurally distinct regions, a larger N-terminal domain containing the active site and a smaller C-terminal domain with other important functions, such as binding to lipids, LDL receptorlike protein, and HSPG (5, 6).Catalytically active LPL is a non-covalent, homodimeric glycoprotein (7, 8). In the rough endoplasmic reticulum (ER), human LPL is co-translationally glycosylated at amino acid residues Asn 43 and Asn 359 . Studies using site-directed mutagenesis and inhibitors of transport between ER and Golgi have shown that core glycosylation at residue Asn 43 is necessary for activation and release of LPL from the ER, but glycosylation at Asn 359 is not necessary (9 -11). A number of studies...

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“…As early as 1994, the protein biosynthesis pathway of the insect cells has been engineered to improve solubility, assembly and secretion of recombinant Ig. 24 In B-cells, the chaperone BiP, a member of Hsp70 family, was first identified in association with the H chain polypeptide in the ER. This protein may have an essential role at preventing the aggregation of the H chain and by maintaining a specific conformation, until final folding in presence of light chain takes place.…”

Section: 69mentioning

confidence: 99%

“…(1) using alternatives promoters, such as earlier viral 9 or cellular promoters, 65,66 (2) modifying or exchanging the signal peptide sequence 67 (3) co-expressing key proteins implicated in the secretion machinery (e.g., chaperone proteins), 9,24,25 and (4) generating stably-transformed insect cells. 65 Comparable experiments were conducted in order to increase the secretion of recombinant antibodies.…”

Section: Enhancing the Production And Secretion Of Recombinant Antibomentioning

confidence: 99%

“…Several immunoglobulin isotypes from many different species, including murine IgG, 2,3,[21][22][23][24][25] chimeric IgG, [26][27][28][29][30] human IgG, [31][32][33][34][35][36][37][38][39] IgA, [40][41][42] IgM 41 and IgE, [43][44][45] as well as pig Igs 30,45 have been successfully expressed in insect cells. These molecules are all correctly processed (signal peptide cleaved), glycosylated and assembled (H2L2) with a production rate varying from 0.75 to 100 mg/l, depending on the antibody ( …”

Section: Introductionmentioning

confidence: 99%

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2012

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Effects of Co-Expressing Chaperone BiP on Functional Antibody Production in the Baculovirus System

Cited by 60 publications

References 0 publications

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha

Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

Lepidopteran cells, an alternative for the production of recombinant antibodies?

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