Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells

Abstract

A protocol is described for the production of both intracellularly expressed and secreted selenomethionyl-derivatized recombinant proteins in baculovirus-infected insect cells. The method results in the production of recombinant soluble proteins with an SeMet occupancy of approximately 75% and with a recovery of approximately 20% that of native protein expression. The method is independent of the percentage methionine content of the protein and is reliable and consistent. Similar results are obtained using either Spodoptera frugiperda Sf9 or Trichoplusia ni High Five insect cells as the expression host, and when cultures are grown in either shake flasks or in Wave BioReactors.

Figure 1.

Effect of time of addition of SeMet on SeMet incorporation into BEVS-produced recombinant FGFR2 kinase domain. High Five insect cells (0.5 L in 1-L shake flasks) were infected with a recombinant baculovirus overproducing the kinase domain of FGFR2 at an moi of ∼1 in ESF921 protein-free medium. After 16 h (A) or 8 h (B) the medium was replaced with ESF921 protein-free, methionine-free medium, and after a further 8 h, SeMet was added to a final concentration of 250 mg/L. Cells were harvested 48 h post-infection and the recombinant FGFR2 isolated from the cell pellet by IMAC chromatography. The His tag was removed by treatment with TEV protease and the protein further purified by reverse IMAC. The purified proteins were analyzed by mass spectrometry.

Figure 2.

Effect of SeMet concentration on SeMet incorporation and on recombinant protein recovery in BEVS-produced recombinant FGFR2 kinase domain. Sf9 insect cells (0.5 L in 1-L shake flasks) were infected with a recombinant baculovirus overproducing the kinase domain of FGFR2 at a moi of ∼1 in ESF921 protein-free medium. After 8 h the medium was replaced with ESF921 protein-free methionine-free medium, and after a further 8 h, SeMet was added to various final concentrations. Cells were harvested 48 h post-infection and the recombinant FGFR2 isolated from the cell pellet by IMAC chromatography. (A) SDS-PAGE analysis of material eluted from IMAC chromatography after expression in the presence of the indicated concentrations of SeMet. (B) Mass spectrogram traces of purified detagged FGFR2 kinase domains following expression in the presence of (A) 0, (B) 50, (C) 100, and (D) 200 mg/L SeMet. The % SeMet incorporation is indicated on the right in each trace (estimated by inspection as described in the Materials and Methods section). (C) Plot of % SeMet incorporation into recombinant FGFR2 kinase domain as a function of the concentration of SeMet used in the growth medium.

Figure 3.

SDS-PAGE analysis of a selection of SeMet-derivatized proteins produced in the BEVS. SeMet-derivatized proteins were produced by infecting High Five insect cells (1 L in 3-L shake flasks) with recombinant baculoviruses overproducing various intracellularly expressed human protein kinase domains, or secreted DP4 peptidase, at a moi of ∼1 in ESF921 protein-free medium. After 8 h the medium was replaced with ESF921 protein-free methionine-free medium, and after a further 8 h, SeMet was added to a final concentration of 250 mg/L. The cells were harvested 48 h post-infection and the recombinant proteins isolated by IMAC chromatography. Native proteins were produced under identical conditions with the exception that SeMet was not added and ESF-921 protein-free medium was used throughout. The figure shows a comparison, by SDS-PAGE analysis, of the recoveries of the native proteins and their SeMet-derivatized counterparts as eluted from the IMAC chromatography columns.

Figure 4.

Mass spectroscopy analysis of a selection of SeMet-derivatized proteins produced in the BEVS. Shown are the mass spectroscopy traces of the proteins produced as described in the legend to ▶. Prior to the analysis, the proteins were further purified by removing the polyhistidine purification tags with TEV protease and repurifying over IMAC chromatography.