Molecular Biotechnology

, Volume 60, Issue 1, pp 31–40 | Cite as

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

  • Le T. M. Le
  • Jens R. Nyengaard
  • Monika M. Golas
  • Bjoern Sander
Original Paper


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.


Recombinant protein expression Signal peptide CD64 FLAG tag Strep tag Baculovirus 



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



E. coli

Escherichia coli


Endoplasmic reticulum


Fetal bovine serum


High-affinity immunoglobulin E receptor γ subunit


Immunoglobulin G


Isopropyl β-d-1-thiogalactopyranoside


Kilo Dalton


Multiple cloning site






Ribosomal nascent chain complexes


Sodium dodecyl sulfate–polyacrylamide gel electrophoresis


Spodoptera frugiperda 21


Spodoptera frugiperda 9


Signal recognition particle





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.

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interests related to this work.

Ethical approval

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

Supplementary material

12033_2017_41_MOESM1_ESM.docx (102 kb)
Supplementary material 1 (DOCX 103 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Le T. M. Le
    • 1
  • Jens R. Nyengaard
    • 1
    • 2
    • 3
  • Monika M. Golas
    • 4
    • 5
  • Bjoern Sander
    • 1
    • 3
    • 6
  1. 1.Stereology and EM Laboratory, Department of Clinical MedicineAarhus UniversityAarhus CDenmark
  2. 2.Core Center for Molecular Morphology, Department of Clinical MedicineAarhus University HospitalAarhus CDenmark
  3. 3.Centre for Stochastic Geometry and Advanced BioimagingAarhus UniversityAarhus CDenmark
  4. 4.Department of BiomedicineAarhus UniversityAarhus CDenmark
  5. 5.Department of Human GeneticsHannover Medical SchoolHannoverGermany
  6. 6.Institute of PathologyHannover Medical SchoolHannoverGermany

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