Vectors for Expression of Signal Peptide-Dependent Proteins in Baculovirus/Insect Cell Systems and Their Application to Expression and Purification of the High-Affinity Immunoglobulin Gamma Fc Receptor I in Complex with Its Gamma Chain

Abstract

Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER). The vectors code for N-terminal and C-terminal affinity tags including 3 × FLAG and Twin-Strep, which represent tags compatible with efficient translocation to the ER as well as with purification under mild conditions that preserve protein structure and function. As a model, we used our system to express and purify the engineered high-affinity immunoglobulin gamma Fc receptor I (CD64) in complex with its gamma subunit (γ-chain). We demonstrate that CD64 expressed in complex with the γ-chain is functional in immunoglobulin G (IgG) binding. The sedimentation of CD64 in complex with IgG suggests individual CD64/IgG complexes in addition to formation of high-molecular weight complexes. In summary, our vectors can be used as a tool for expression of membrane proteins, other secretory pathway proteins and their protein complexes.

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Abbreviations

CD64:

Cluster of differentiation 64; high-affinity immunoglobulin gamma Fc receptor I

C-terminal:

Carboxy-terminal

E. coli :

Escherichia coli

ER:

Endoplasmic reticulum

FBS:

Fetal bovine serum

γ-chain:

High-affinity immunoglobulin E receptor γ subunit

IgG:

Immunoglobulin G

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

kDa:

Kilo Dalton

MCS:

Multiple cloning site

N-terminal:

Amino-terminal

P/S:

Penicillin/streptomycin

RNCs:

Ribosomal nascent chain complexes

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Sf21:

Spodoptera frugiperda 21

Sf9:

Spodoptera frugiperda 9

SRP:

Signal recognition particle

X-gal:

5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside

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Acknowledgements

We thank Golshah Ayoubi and Susanne N. Stubbe for expert technical assistance, and Srdja Drakulic for initial help with experiments. The work has been supported by a grant from the Danish Council for Independent Research for Natural Sciences, a grant from the A.P. Møller Foundation for the Advancement of Medical Science and a grant from the Fabrikant Einar Willumsen Mindelegat to B.S. The Centre for Stochastic Geometry and Advanced Bioimaging is supported by the Villum Foundation. M.M.G. acknowledges support from the Lundbeck Foundation and the Carlsberg Foundation.

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Correspondence to Bjoern Sander.

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The study does not use human material, patient data or animals.

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Le, L.T.M., Nyengaard, J.R., Golas, M.M. et al. Vectors for Expression of Signal Peptide-Dependent Proteins in Baculovirus/Insect Cell Systems and Their Application to Expression and Purification of the High-Affinity Immunoglobulin Gamma Fc Receptor I in Complex with Its Gamma Chain. Mol Biotechnol 60, 31–40 (2018). https://doi.org/10.1007/s12033-017-0041-8

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Keywords

  • Recombinant protein expression
  • Signal peptide
  • CD64
  • FLAG tag
  • Strep tag
  • Baculovirus