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Production of Phosphorylated Ric-8A proteins using protein kinase CK2.

Abstract：

:Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Yu W,Yu M,Papasergi-Scott MM,Tall GG

doi

10.1016/j.pep.2018.10.002

subject

Has Abstract

pub_date

2019-02-01 00:00:00

pages

98-103

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(18)30369-3

journal_volume

154

pub_type

杂志文章

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