Enhanced Protein Secretion From Insect Cells by Co-Expression of the Chaperone Calreticulin and Translation Initiation Factor eIF4E

Teng, Chao-Yi; Chang, Shou-Lin; Oers, Monique M. van; Wu, Tzong‐Yuan

doi:10.1007/s12033-012-9545-4

Cited by 9 publications

(9 citation statements)

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“…Colocalization and immunoprecipitation experiments indicated that in uninfected mosquito cells, CAV-1 is indeed associated with all 3 chaperones proposed to form the complex, FKPB52, Cy40, and CyA; moreover, knocking down the expression of CAV-1 (data not shown) or any of the associated chaperones resulted in a significant decrease in the lipid droplet count per cell, indicating a role for the CCC in lipid homeostasis in mosquito cells. In agreement with our results, ER chaperones have been reported to be necessary for the secretion of recombinant proteins expressed in insect cells using a baculovirus system (30,31).…”

Section: Discussionsupporting

confidence: 93%

The Dengue Virus Nonstructural Protein 1 (NS1) Is Secreted from Mosquito Cells in Association with the Intracellular Cholesterol Transporter Chaperone Caveolin Complex

Rosales

Ludert

2019

J Virol

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Dengue virus (DENV) is a mosquito-borne virus of the family Flaviviridae. The RNA viral genome encodes three structural and seven nonstructural proteins. Nonstructural protein 1 (NS1) is a multifunctional protein actively secreted in vertebrate and mosquito cells during infection. In mosquito cells, NS1 is secreted in a caveolin-1-dependent manner by an unconventional route. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40, and CyA and is responsible for the cholesterol traffic inside the cell. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 associates with and relies on the CCC for secretion. Treatment of mosquito cells with classic secretion inhibitors, such as brefeldin A, Golgicide A, and Fli-06, showed no effect on NS1 secretion but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV-1 or FKBP52 with short interfering RNAs or the inhibition of CyA by cyclosporine resulted in significant decrease in NS1 secretion, again without affecting virion release. Colocalization, coimmunoprecipitation, and proximity ligation assays indicated that NS1 colocalizes and interacts with all proteins of the CCC. In addition, CAV-1 and FKBP52 expression was found augmented in DENV-infected cells. Results obtained with Zika virus-infected cells suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway as that observed for DENV NS1. These results uncover important differences in the dengue virus-cell interactions between the vertebrate host and the mosquito vector as well as novel functions for the chaperone caveolin complex. IMPORTANCE The dengue virus protein NS1 is secreted efficiently from both infected vertebrate and mosquito cells. Previously, our group reported that NS1 secretion in mosquito cells follows an unconventional secretion pathway dependent on caveolin-1. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 secretion takes place in association with the chaperone caveolin complex, a complex formed by caveolin-1 and the chaperones FKBP52, CyA, and Cy40, which are in charge of cholesterol transport inside the cell. Results obtained with ZIKV-infected mosquito cells suggest that ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. These results uncover important differences in the virus-cell interactions between the vertebrate host and the mosquito vector, as well as novel functions for the chaperone caveolin complex. Moreover, manipulation of the NS1 secretory route may prove a valuable strategy to combat these two mosquito-borne diseases. Volume 93 Issue 4 e01985-18 jvi.asm.org 2 on March 18, 2020 by guest http://jvi.asm.org/ Downloaded from plasma membrane, mediated by the CCC, to reach the extracellular space. In addition, data are presented suggesting that a similar pathway is used for the secretion of Zika virus NS1 protein in infected m...

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Section: Discussionsupporting

confidence: 93%

The Dengue Virus Nonstructural Protein 1 (NS1) Is Secreted from Mosquito Cells in Association with the Intracellular Cholesterol Transporter Chaperone Caveolin Complex

Rosales

Ludert

2019

J Virol

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“…Gantke et al co-expressed the Ebola virus protein 35, which is a viral protein that prevents translational silencing, and increased recombinant protein production by 10-fold [16]. An alternative approach to circumvent translational silencing in insect cells following baculovirus infection is to co-express eIF4E, which resulted in a 2-fold increase in the production of a secreted alkaline phosphatase (SEAP)-EGFP fusion protein (SEFP)[17]. …”

Section: Optimizing Transcription and Enhancing Translationmentioning

confidence: 99%

“…In insect cells, host protein biosynthesis shuts down as a result of infection by the recombinant baculovirus, which can adversely affect levels of molecular chaperones important for the folding of secreted proteins and membrane proteins in the endoplasmic reticulum (ER), particularly in relation to the high levels of protein synthesis resulting from high mRNA levels produced from the polyhedrin promoter. Hence, co-expression of the membrane-bound molecular chaperone calnexin enhanced the expression of functional serotonin transporter (SERT) by nearly 3 fold [21], and co-expression of the soluble molecular chaperone calreticulin increased secretion of SEFP in insect cells [17]. Whether a lack of appropriate molecular chaperones in heterologous systems contributes to low levels of functional protein sometimes is difficult to assess.…”

Section: Folding and Secretory Pathway Engineeringmentioning

confidence: 99%

“…Part of the problem is that overexpression of ER resident chaperones such as calreticulin might burden the ER and activate an unfolded protein response [17]. Another more challenging issue is that molecular chaperones may act in a concerted fashion to promote protein folding in a poorly understood process, suggesting that it may be best to overexpress multiple chaperones simultaneously.…”

Section: Folding and Secretory Pathway Engineeringmentioning

confidence: 99%

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Engineering cells to improve protein expression

Xiao

Shiloach

Betenbaugh

2014

Current Opinion in Structural Biology

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Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes.

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“…Moreover, this shutdown leads to molecular chaperone and foldase insufficiencies in the ER, which in turn result in protein aggregation (Fath-Goodin et al, 2006;Teng et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Effect of a Bombyx mori Protein Disulfide Isomerase on Production of Recombinant Antibacterial Peptides

Goo¹,

Kim²,

Choi³

et al. 2013

International Journal of Industrial Entomology

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The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins. IntroductionThe production of recombinant proteins from cultured insect cells for use in diagnostics, protein and biomedical research, and as vaccines is increasing, and currently is a major Turano et al., 2002;Wilkinson and Gilbert, 2004). We previously isolated Bombyx mori protein disulfide isomerase (bPDI), which has two thiol oxidoreductase sites and enzymatic activity for reduced and scrambled RNase, like other PDI family members (Goo et al., 2002;Goo et al., 2008

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Enhanced Protein Secretion From Insect Cells by Co-Expression of the Chaperone Calreticulin and Translation Initiation Factor eIF4E

Cited by 9 publications

References 38 publications

The Dengue Virus Nonstructural Protein 1 (NS1) Is Secreted from Mosquito Cells in Association with the Intracellular Cholesterol Transporter Chaperone Caveolin Complex

The Dengue Virus Nonstructural Protein 1 (NS1) Is Secreted from Mosquito Cells in Association with the Intracellular Cholesterol Transporter Chaperone Caveolin Complex

Engineering cells to improve protein expression

Effect of a Bombyx mori Protein Disulfide Isomerase on Production of Recombinant Antibacterial Peptides

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