Abstract
The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins.
Highlights
Improved yields and purity of LRR proteins and multiprotein complexes through chaperone co-expression, DNA construct design, and use of polycistronic baculovirus.
Over 300-fold yield increase of SMP protein complex from 0.1 mg/L to 32.6 mg/L.
SUGT1 co-expression is a highly effective technique for recombinant LRR protein expression and purification.
Polycistronic baculovirus infection is ideal for production of multiprotein complexes
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- CV
- column volume
- IMAC
- immobilized metal ion affinity chromatography
- LRR
- leucine-rich repeat
- MWCO
- molecular weight cut-off
- SDS-PAGE
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SEC
- size exclusion chromatography
- SMP
- SHOC2/MRAS/PPP1CA protein complex
- TEV
- tobacco etch virus proteins