Effective production of oligomeric membrane proteins by EarlyBac-insect cell system

Abstract

Despite major advances in methodologies for membrane protein production over the last two decades, there remain challenging protein complexes that are technically difficult to yield by conventional recombinant expression methods. A large number of these proteins are multimeric membrane proteins from eukaryotic species, which are required to pass through stringent quality control mechanisms of host cells for proper folding and complex assembly. Here, we describe the development procedure to improve the production efficiency of multi-oligomeric membrane protein complexes in insect cells and recombinant baculovirus, which involves screening of promoters, enhancers, and untranslated regions for expression levels, using calcium homeostasis modulator (CALHM) and N-methyl-d-aspartate receptor (NMDAR) proteins as examples. We demonstrate that our insect cell expression strategy is effective in expression of both multi-homomeric CALHM proteins and multi-heteromeric NMDARs.

Keywords: CALHM; Channel; Cryo-EM; EarlyBac; Expression system; Membrane proteins; NMDA receptors; Oligomeric proteins; X-ray crystallography and; ab-FSEC.

Fig. 1.. Expression assessment in insect cell/baculovirus.

(A) GluN1 was C-terminally fused to EGFP and the cytoplasmic domain of GluN2B was truncated (GluN2Bct) (Karakas and Furukawa, 2014). The GluN1-EGFP and GluN2Bct were inserted into pUCDM and pFL vectors, respectively, and recombined using Cre-lox recombination. Baculovirus was made using the Bac-to-Bac system (ThermoFisher). (B) Sf9 cells in a 6-well plate were infected with the recombinant baculovirus. Cells were solubilized and subjected to FSEC (475 nm excitation and 507 nm emission). (C) FSEC peaks of GluN1-EGFP/GluN2Bct NMDAR where hetero-tetrameric peak (arrow) was observed more prominently under CMV promoter (red trace) than polyhedrin promoter (polH; blue trace).

Fig. 2.. Screening of cis elements for expression.

(A) Various promoters were screened (Screen 1) for expression of GluN1-EGFP/GluN2Bct NMDAR. FSEC showed the highest protein expression under dHsp70 promoter (arrow). (B) Enhancers (Screen 2), 3’ UTRs (Screen 3), and 5’ UTRs (Screen 4) were screened using EGFP-CALHM2. The FSEC analysis indicated maximized expression when a combination of hr1 or hr5 and p10 3’UTR is used.

Fig. 3.. Modified FSEC using 1D4 antibody.

(A) Schematics of the modified FSEC using FITC labelled 1D4 antibodies (ab-FSEC). (B) Representative FSEC trace of 1D4 tagged (C-terminus of GluN2Bct) NMDARs. Arrow1 and 2 indicate NMDARs-FITC-1D4 and free FITC-1D4, respectively. Excitation and emission wavelengths are 494 nm and 521 nm, respectively.

Fig. 4.. Expression and purification of NMDAR.

(A) The Earlybac expression vectors, pUCp10 and pFp10. GluN1 and GluN2Bct are in pUCp10 and pFp10, respectively. Dual Strep-Tactin tag (DS) is in GluN2Bct after the signal peptide sequence. (B) Flow chart of purification (left panel) and representative SDS-PAGE (10%, middle panel), and FSEC detecting tryptophan fluorescence (excitation/emission = 280 nm/330 nm; right panel).