Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinantleucine-rich repeat proteins and protein complexes

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. HighlightsO_LIImproved yields and purity of LRR proteins and multiprotein complexes through chaperone co-expression, DNA construct design, and use of polycistronic baculovirus. C_LIO_LIOver 300-fold yield increase of SMP protein complex from 0.1 mg/L to 32.6 mg/L. C_LIO_LISUGT1 co-expression is a highly effective technique for recombinant LRR protein expression and purification. C_LIO_LIPolycistronic baculovirus infection is ideal for production of multiprotein complexes C_LI