Improved Production of Class I Phosphatidylinositol 4,5-bisphosphate 3-kinase

Author:

Messing,Simon

Widmeyer, Stephanie R T

Denson,John-Paul

Mehalko,Jennifer

Wall,Vanessa

Drew,Matt

Snead,Kelly

Hong,Min

Grose,Carissa

Esposito,Dom

Gillette,Bill
Author Address

Protein Expression Laboratory, NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA. Electronic address: simon.messing@nih.gov.,

Abstract:

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110a/p85a and a 3-fold increase in p110a. We also used these protocols to produce comparable constructs of the ß and d isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases. Copyright © 2024. Published by Elsevier Inc.

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