Efficient production of protein complexes in mammalian cells using a poxvirus vector

Abstract

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10–50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro

“…2 C ) produced as a complex in BHK21 cells (see purification gel in Fig. S1 A ), having undergone the post-translational modifications (PTM) that could be crucial for the nucleation of LLPS ( 37 , 38 ). As expected, both IN-LEDGF/p75 complexes showed an ability to form LLPS in the presence of PEG-4000 in vitro ( Fig.…”

“…BHK21 overexpression with MVA expression vector. We used a vaccinia virus strain (Modified Vaccinia Ankara - MVA - Bio safety level 1 - Table 1 ) allowing an inducible expression of the IN-LEDGF/p75 complex in BHK21 mammalian cells ( 37 , 38 ). Briefly, the polygene of interest (coding for the two proteins IN and 6His-LEDGF/p75) was integrated by homologous recombination at the HA locus of the MVA viral genome and the recombinant viruses were selected using a resistance marker cassette present on the polygene as described in ( 37 , 38 ).…”

“…We used a vaccinia virus strain (Modified Vaccinia Ankara - MVA - Bio safety level 1 - Table 1 ) allowing an inducible expression of the IN-LEDGF/p75 complex in BHK21 mammalian cells ( 37 , 38 ). Briefly, the polygene of interest (coding for the two proteins IN and 6His-LEDGF/p75) was integrated by homologous recombination at the HA locus of the MVA viral genome and the recombinant viruses were selected using a resistance marker cassette present on the polygene as described in ( 37 , 38 ). BHK21 C13-2P suspension cells ( Table 1 ) were grown in Glasgow's modified Eagle’s medium (GMEM; Thermo Fisher) supplemented with Bacto Tryptose Phosphate (1.5 g/L), 10% fetal calf serum and 50 μM gentamycin.…”