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. 2018 Jan;60(1):31-40. doi: 10.1007/s12033-017-0041-8.

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.

Keywords: Baculovirus; CD64; FLAG tag; Recombinant protein expression; Signal peptide; Strep tag.

MeSH terms

  • Animals
  • Baculoviridae / genetics*
  • Baculoviridae / metabolism
  • Genetic Vectors*
  • Humans
  • Immunoglobulin G / genetics
  • Immunoglobulin G / metabolism
  • Insecta / cytology*
  • Insecta / genetics
  • Protein Engineering / methods
  • Protein Sorting Signals / genetics
  • Receptors, IgG / genetics*
  • Receptors, IgG / metabolism
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Immunoglobulin G
  • Protein Sorting Signals
  • Receptors, IgG
  • Recombinant Proteins