Molecular Chaperone-Assisted Production of Human α-1,4-N-Acetylglucosaminyltransferase in Silkworm Larvae Using Recombinant BmNPV Bacmids

Nakajima, Makoto; Kato, Tatsuya; Kanamasa, Shin; Park, Enoch Y.

doi:10.1007/s12033-009-9174-8

Cited by 11 publications

(8 citation statements)

References 25 publications

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“…Bacmids allow easy coexpression of a chaperone with a target protein. The expression level of a recombinant GFP uv -human α1,4- N -acetylglucosaminyltransferase (α4GnT) fusion protein was enhanced in silkworm larvae, by coexpression of a human molecular chaperone, human calnexin (CNX), or immunoglobulin heavy-chain binding protein (BiP; Nakajima et al 2009). The expression of CNX and BiP under the control of the polyhedrin promoter enhanced the α4GnT activity by 1.4- and 2.0-fold, respectively.…”

Section: Improved Protein Expression By a Modified Bmnpv Bacmidmentioning

confidence: 99%

Silkworm expression system as a platform technology in life science

Kato

Kajikawa

Maenaka

et al. 2009

Appl Microbiol Biotechnol

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178

110

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Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system.

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Section: Improved Protein Expression By a Modified Bmnpv Bacmidmentioning

confidence: 99%

Silkworm expression system as a platform technology in life science

Kato

Kajikawa

Maenaka

et al. 2009

Appl Microbiol Biotechnol

Self Cite

178

110

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“…Therefore, we assumed that this is a key explanation for baculovirus-infected Sf -caspase-1-repressed stable cells have a higher recombinant protein production than that in normal cells. The persistent expression of molecular chaperones in baculovirus-infected Sf -caspase-1-repressed stable cells resulted in a higher recombinant protein production than that in normal cells, which can be suggested by some earlier studies that focused on the co-expression of molecular chaperones, Bip (GRP78), Calreticulin and Calnexin in BEVS to improve the recombinant production and secretion from cells and larvas [35-38]. Therefore, we also suppose that expression of molecular chaperon in combined with RNAi technique may have opportunity to further improve BEVS.…”

Section: Discussionmentioning

confidence: 99%

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

et al. 2012

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BackgroundThere are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated.ResultsIn the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.ConclusionThe data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.

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“…This indicates that chaperone overexpression is not necessary for the enhancement of recombinant protein production. Indeed, in silkworm larvae, the expression of CNX or Bip under the control of the ie-2 promoter, which has lower activity than that of the polyhedrin promoter, is sufficient to enhance the production of the GFP uv -␣1,4-N-acetylglucosaminyltransferase fusion protein (Nakajima et al, 2009). Various chaperones with moderate expression levels exist in the ER, and together, they assist with protein folding.…”

Section: Discussionmentioning

confidence: 99%

New strategy for rapid isolation of stable cell lines from DNA-transformed insect cells using fluorescence activated cell-sorting

Kato

Yoshizuka

Park

2010

Journal of Biotechnology

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A stably transformed insect cell expression system is superior to a baculovirus expression system, since the expression system is sustained and there is no cell lysis, but the isolation of cell lines producing recombinant proteins is time-consuming and laborious. In this study, we developed a technique for the rapid isolation and efficient cultivation of sorted cells in a 24 well deep plate Bioshaker, utilizing the fluorescence activated cell-sorting (FACS) method. TnpXme11 cells, which stably expressed GFP(uv)-beta1,3-N-acetylglucosaminyltransferase2 (GGT2), were transfected with a plasmid vector for the expression of a molecular chaperone (TnpXme11-hCNX6 cell line). The expression levels of GGT2 and the molecular chaperone fused with HcRed were analyzed by FACS. Two cell lines were established by single and double sorting. Sorting of the top 10% of the TnpXme11-hCNX6 cell population with the highest fluorescence yielded the TnpXme11-hCNX6-1 cell line. TnpXme11-hCNX6-2 cells were created in a similar fashion, as mentioned above, by a second sorting of the top 10% of the TnpXme11-hCNX6-1 cell population with the highest fluorescence. The cells thus isolated produced approximately 2-fold higher extracellular activity than that before cell sorting. This procedure can be accomplished in only 2 weeks, including transfection, isolation and analysis of high protein-producing cells, and is a breakthrough strategy for the rapid isolation of a recombinant, stable insect cell line.

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Molecular Chaperone-Assisted Production of Human α-1,4-N-Acetylglucosaminyltransferase in Silkworm Larvae Using Recombinant BmNPV Bacmids

Cited by 11 publications

References 25 publications

Silkworm expression system as a platform technology in life science

Silkworm expression system as a platform technology in life science

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

New strategy for rapid isolation of stable cell lines from DNA-transformed insect cells using fluorescence activated cell-sorting

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