SweetBac: A New Approach for the Production of Mammalianised Glycoproteins in Insect Cells

Dieter Palmberger,Imre Berger,Iain B. H. Wilson,Reingard Grabherr,Dubravko Rendic,Yi Li

doi:10.1371/journal.pone.0034226

Abstract

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine β1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells.

Highlights

The use of insect cells for the production of therapeutically active proteins has gained increasing importance over the last decade, highlighted by the market entry of the human papilloma virus vaccine CervarixTM [1]
Our own data further indicate that the invertebrate GLY-20 N-acetylglucosaminyltransferase II (GnTII) of C. elegans, whose temperature optimum is near to that where insect cells are grown, was well expressed in Sf9 cells (Wang H., Rendic D., unpublished data)
We constructed a glyco-module coding for a C. elegans GnTII and a bovine galactosyltransferase I (GalT) controlled by p10 and polyhedrin promoter respectively

Summary

Introduction

The use of insect cells for the production of therapeutically active proteins has gained increasing importance over the last decade, highlighted by the market entry of the human papilloma virus vaccine CervarixTM [1]. A set of different insect cell lines, suitable for the baculovirus-driven production of recombinant proteins, is available. A new Trichoplusia ni cell line, BTI-Tnao, has been established [5]. A major limitation regarding the production of therapeutic proteins in insect cell lines is the lack of complex type N-glycans, often leading to a severely reduced efficacy. N-glycans found on insect cell expressed proteins are mainly of a high mannose type or non-fucosylated and core-fucosylated trimannose structures [6]

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