Expression and purification of active human kinases using Pichia pastoris as a general-purpose host

The heterologous expression of human kinases in good purity and a monomeric, soluble active form can be challenging. Most the reported successful attempts are carried out insect cells as host. use E. coli for is limited to few usually facilitated by large solubility tags that limit biophysical studies affect protein–protein interactions. In this report, we evaluate methylotrophic yeast Pichia pastoris (P. pastoris) general-purpose host kinases. Six diverse were chosen due their therapeutic importance cancers. Tested proteins include serine/threonine cyclin-dependent 4 6 (CDK4 6) aurora kinase A (AurKA), receptor tyrosine erbB-2 (HER2), dual specificity mitogen-activated protein 3 (MKK3b). Noting positively charged expressed with higher yield, sought improve two challenging targets, CDK6 HER2, fusing highly basic, N-terminal domain secreted tyrosine-protein VLK. standard procedure P. was adopted, followed purification using affinity chromatography. Purity activity confirmed compared published values. Some purified yield comparable commercially available versions. Addition VLK improved decreased aggregation HER2.