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J Struct Biol. 2018 Aug;203(2):71-80. doi: 10.1016/j.jsb.2018.03.004. Epub 2018 Mar 12.

Baculovirus-driven protein expression in insect cells: A benchmarking study.

Author information

1
Vienna Biocenter Core Facilities GmbH, Dr. Bohrgasse 3, 1030 Vienna, Austria.
2
Max-Planck Institute of Biochemistry, Structural Cell Biology Department, Am Klopferspitz 18, 82152 Martinsried, Germany.
3
Lead Discovery Center GmbH, Otto-Hahn-Str. 15, 44227 Dortmund, Germany.
4
European Molecular Biology Laboratory (EMBL) Heidelberg, Protein Expression and Purification Core Facility, 69117 Heidelberg, Germany.
5
NKI Protein Facility, Division of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
6
Protein Expression Facility, The University of Queensland, Brisbane, Queensland 4072, Australia.
7
Protein Expression Purification and Characterization Facility, Max Planck Institute of Molecular Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, D-01307 Dresden, Germany.
8
Helmholtz Center Munich, Protein Expression and Purification Facility, Institute of Structural Biology, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
9
Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Trieste 34149, Italy.
10
Helmholtz Centre for Infection Research, Department Structure and Function of Proteins, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
11
Lund University, Lund Protein Production Platform (LP3), Sölvegatan 35, SE-223 62 Lund, Sweden.
12
Novartis Institutes for BioMedical Research, Emeryville, CA, USA.
13
Max Planck Institute of Molecular Physiology, Dortmund Protein Facility, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
14
Max-Planck Institute of Biochemistry, Biochemistry Core Facility, Am Klopferspitz 18, 82152 Martinsried, Germany.
15
Coriolis Pharma, Biopharmaceutical Research and Development Service, Am Klopferspitz 19, 82152 Martinsried, Germany.
16
Protein Expression Facility, The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: l.lua@uq.edu.au.
17
Max-Planck Institute of Biochemistry, Biochemistry Core Facility, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: suppmann@biochem.mpg.de.

Abstract

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.

KEYWORDS:

Baculovirus-insect cell system; Benchmark

PMID:
29545204
DOI:
10.1016/j.jsb.2018.03.004

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