Production and Isolation of Magnetic Protein Crystals in HEK293T Cells

Thomas L Li; Bianxiao Cui

doi:10.21769/BioProtoc.3684

Production and Isolation of Magnetic Protein Crystals in HEK293T Cells

Bio Protoc. 2020 Jul 20;10(14):e3684. doi: 10.21769/BioProtoc.3684.

Authors

Thomas L Li¹, Bianxiao Cui¹

Affiliation

¹ Department of Chemistry, Stanford University, Stanford, California, USA.

Abstract

Advances in protein engineering have enabled the production of self-assembled protein crystals within living cells. Our recent publication demonstrates the production of ftn-PAK4, which is a ferritin-containing crystal that can mineralize iron and become magnetic when isolated. We have developed an optimized protocol for the production and isolation of PAK4-based crystals. The crystals are first grown in low-passage HEK293T cells, released using a lysis buffer containing NP-40 and DNase, and collected under careful centrifugation conditions. Our protocol maximizes the purity and yield of crystals and is quick and straightforward.

Keywords: Crystal; Isolation; Protein Crystal; ftn-PAK4; “In cellulo”.

Grants and funding

R01 GM125737/GM/NIGMS NIH HHS/United States