Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells.

Human 90K (h90K; Mac-2-binding protein) glycoprotein is a potential pharmaceutical due to its inhibitory activity against cancer metastasis and expansion. Here, h90K glycoprotein was produced in insect Drosophila S2 cell system, and its N-glycan pattern was analyzed. A plasmid encoding h90K gene, fused with a hexahistidine tag under the control of Drosophila metallotionein promoter, was stably transfected into S2 cells. After copper sulfate induction, transfected S2 cells secreted recombinant h90K at a good expression level of 28mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of ∼78kDa which was much smaller than that (∼97kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at ∼60kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (∼18kDa) of N-glycans of the natural h90K. Through detail analyses using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the S2-derived recombinant h90K was confirmed that it has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major (∼79%) N-glycans.

In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells.

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.

Expression of ß-1,4-galactosyltransferase and suppression of ß-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells.

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of ß-N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of ß-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

Recombinant mussel adhesive protein as a gene delivery material.

Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant mussel adhesive protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine 2000. Our results indicate that this mussel adhesive protein may be used as a potential protein-based gene-transfer mediator.

Suppression of beta-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells.

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. Previously, we have shown that paucimannosidic N-glycan structures are dominant in Drosophila S2 cells. It has been proposed that beta-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation in several insects and insect-derived cells except S2 cells. In the present work, we investigated the substantial effects of GlcNAcase on N-glycan patterns in Drosophila S2 cells using two GlcNAcase suppression strategies: an mRNA-targeting approach using RNA interference (RNAi) and a protein-targeting approach using the specific chemical inhibitor 2-acetamido-1,2-dideoxynojirimycin (2-ADN). Using high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses, we found that the N-glycosylation patterns of human erythropoietin (hEPO) secreted by stably transfected S2 cells were more complex following GlcNAcase suppression, which generated N-glycan structures with a terminal GlcNAc and/or galactose. These data demonstrate that GlcNAcase may be an important factor in the formation of paucimannosidic core N-glycans in Drosophila S2 cells and suggest that it may be possible to express complex glycoproteins in engineered Drosophila S2 cells by suppressing GlcNAcase in the N-glycosylation pathway.

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture.

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.

Statistical determination of optimal baculovirus infection condition for recombinant protein production in Drosophila S2 cells.

Insect Drosophila melanogaster S2 cell was developed as plasmid-based and, therefore, a nonlytic expression system for functional foreign proteins. To achieve multiple protein expressions, it was suggested that baculovirus be used on S2 cell system because baculovirus can infect S2 cells but cannot replicate inside the cells. Therefore, establishment of baculovirus infection conditions is the first important step and this should be properly optimized for production yield. We used statistical methodology to optimize the baculovirus infection conditions using green fluorescent protein (GFP) as a reporter protein. Consequently, we arrived at optimal infection conditions through a statistical regression method. The secreted GFP yield from vMT-GFP baculovirus-infected wild-type S2 cells under optimal infection conditions was >15-fold higher than that under nonoptimal conditions and comparable to that from stably transfected recombinant S2 cells.

Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates.

Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate-biomolecule interactions. In the present work, we report a simple and rapid modification technique for diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose (disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates, direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions and carbohydrate sensor or array development for diagnosis and screening.