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

Snead, Kelly; Wall, Vanessa; Ambrose, Hannah; Esposito, Dominic; Drew, Matthew

doi:10.1016/j.pep.2022.106061

Cited by 9 publications

(4 citation statements)

References 22 publications

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“… 44 Expression of His6-MBP-tev-GG-Hs.KRAS4b (2-188) (for both the polyhedrin promoter and the p10 promoter constructs), His6-MBP-tev-GG-Hs.KRAS4b(2-188) S106C/C118S, and His6-MBP-tev-RAF1(52-188) K148A/K157A/K179A was as described in ref. 49 .…”

Section: Methodsmentioning

confidence: 99%

Membrane lipids drive formation of KRAS4b-RAF1 RBDCRD nanoclusters on the membrane

Shrestha,

Carpenter,

Van

et al. 2024

Commun Biol

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The oncogene RAS, extensively studied for decades, presents persistent gaps in understanding, hindering the development of effective therapeutic strategies due to a lack of precise details on how RAS initiates MAPK signaling with RAF effector proteins at the plasma membrane. Recent advances in X-ray crystallography, cryo-EM, and super-resolution fluorescence microscopy offer structural and spatial insights, yet the molecular mechanisms involving protein-protein and protein-lipid interactions in RAS-mediated signaling require further characterization. This study utilizes single-molecule experimental techniques, nuclear magnetic resonance spectroscopy, and the computational Machine-Learned Modeling Infrastructure (MuMMI) to examine KRAS4b and RAF1 on a biologically relevant lipid bilayer. MuMMI captures long-timescale events while preserving detailed atomic descriptions, providing testable models for experimental validation. Both in vitro and computational studies reveal that RBDCRD binding alters KRAS lateral diffusion on the lipid bilayer, increasing cluster size and decreasing diffusion. RAS and membrane binding cause hydrophobic residues in the CRD region to penetrate the bilayer, stabilizing complexes through β-strand elongation. These cooperative interactions among lipids, KRAS4b, and RAF1 are proposed as essential for forming nanoclusters, potentially a critical step in MAP kinase signal activation.

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Section: Methodsmentioning

confidence: 99%

Membrane lipids drive formation of KRAS4b-RAF1 RBDCRD nanoclusters on the membrane

Shrestha,

Carpenter,

Van

et al. 2024

Commun Biol

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“…The structure of the SMP complex independently determined by four different research groups shows a similar arrangement of three proteins. We co-expressed SHOC2 and the pre-assembled SMP complex using the LRR folding chaperone, SUGT1, to enhance their expression [48,51], while the other three groups purified individual proteins and assembled the complex before purifying it again [22,49,50] A and 2.17 A, respectively [22,[48][49][50]52,53]. Each X-ray structure contained two copies of the SMP complex in the asymmetric unit.…”

Section: Assembly and Structure Of The Smp Complexmentioning

confidence: 99%

“…The SMP complex specifically dephosphorylates CR2-pS of RAF substrates. Other phosphorylation sites such as CR3-pS, pS445 (BRAF) and pS338 (RAF1) are not dephosphorylated by the SMP complex [9,15,48,51]. Although PP1C alone has poor activity towards CR2-pS, high concentrations of PP1C can dephosphorylate CR2-pS [48].…”

Section: Dephosphorylation Of Raf Substratesmentioning

confidence: 99%

Structural insights into the role of SHOC2‐MRAS‐PP1C complex in RAF activation

Bonsor

Simanshu

2023

The FEBS Journal

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RAF activation is a key step for signalling through the mitogen‐activated protein kinase (MAPK) pathway. The SHOC2 protein, along with MRAS and PP1C, forms a high affinity, heterotrimeric holoenzyme that activates RAF kinases by dephosphorylating a specific phosphoserine. Recently, our research, along with that of three other teams, has uncovered valuable structural and functional insights into the SHOC2‐MRAS‐PP1C (SMP) holoenzyme complex. In this structural snapshot, we review SMP complex assembly, the dependency on the bound‐nucleotide state of MRAS, the substitution of MRAS by the canonical RAS proteins and the roles of SHOC2 and MRAS on PP1C activity and specificity. Furthermore, we discuss the effect of several RASopathy mutations identified within the SMP complex and explore potential therapeutic approaches for targeting the SMP complex in RAS/RAF‐driven cancers and RASopathies.

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“…Herein, we focus on insect cell expression and describe a mid‐scale platform used to perform successful expression and purification screens of intracellular multiprotein complexes. Although protein co‐expression using single open reading frame (ORF)‐encoding plasmids is widely used in baculovirus‐mediated insect cell expression, the use of multi‐ORF or polycistronic plasmid vectors offers a significant advantage for reconstituting protein complexes with two or more proteins because this approach uses fewer viruses during co‐expression and also ensures uniform expression in insect cells (Snead et al., 2022). Overexpression of multiple proteins from a single plasmid vector can be achieved either by using individual promoters to drive the transcription of each ORF or by using a polycistronic construct, in which a single mRNA transcript encodes multiple proteins.…”

Section: Introductionmentioning

confidence: 99%

End‐to‐End Semi‐automated Mid‐scale Protein Screening Platform for Drug Discovery Research

Zilberleyb,

Kugel,

Patel

et al. 2023

Current Protocols

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The drug discovery landscape is ever‐evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher‐throughput mid‐scale semi‐automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult‐to‐express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high‐quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co‐expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation‐prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co‐infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA‐encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Baculovirus generation via homologous recombinationSupport Protocol 1: Anti‐glycoprotein 64 antibody assayBasic Protocol 2: Generation of insect cell biomass expressing target protein(s)Basic Protocol 3: Mid‐scale affinity purificationSupport Protocol 2: Automated method for affinity purification on Hamilton STARBasic Protocol 4: Size exclusion chromatographySupport Protocol 3: Chromeleon 7 operation on Vanquish Duo

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

Cited by 9 publications

References 22 publications

Membrane lipids drive formation of KRAS4b-RAF1 RBDCRD nanoclusters on the membrane

Membrane lipids drive formation of KRAS4b-RAF1 RBDCRD nanoclusters on the membrane

Structural insights into the role of SHOC2‐MRAS‐PP1C complex in RAF activation

End‐to‐End Semi‐automated Mid‐scale Protein Screening Platform for Drug Discovery Research

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