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

Production and Isolation of Magnetic Protein Crystals in HEK293T Cells

Thomas L Li  1 , Bianxiao Cui  1
Affiliations

Production and Isolation of Magnetic Protein Crystals in HEK293T Cells

Thomas L Li et al. Bio Protoc. .

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”.

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Conflict of interest statement

Competing interestsThe authors declare that there are no competing interests or conflicts of interest.

Figures

Figure 1.
Figure 1.. HEK293T cells containing ftn-PAK4 and inka-PAK4 crystals, with GFP-PAK4, 72 h after transfection.
These crystals are ready for isolation.
Figure 2.
Figure 2.. A ftn-PAK4 isolation without, and with DNase.
In the DNAse-free condition, many of the crystals have been trapped in a sticky pellet, substantially reducing the concentration of free crystals.
Figure 3.
Figure 3.. Ideal colors for ftn-PAK4 and inka-PAK4 crystals after iron loading and PB stain
See this image and copyright information in PMC

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