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bioRxiv · 10.1101/2022.01.19.476911

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

Snead, K.·Wall, V.·Ambrose, H.·Esposito, D.·Drew, M.
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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. 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

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Snead, K., Wall, V., Ambrose, H., Esposito, D., Drew, M.. 2022-01-20. Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinantleucine-rich repeat proteins and protein complexes. https://doi.org/10.1101/2022.01.19.476911

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