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: Protein Crystal, Isolation, ftn-PAK4, “In cellulo”, Crystal
Background
Recent works have reported the production and isolation of “in cellulo” crystals through the heterologous expression of proteins in living cells. These crystals have varied applications, such as cargo delivery (Ijiri et al., 2009) or x-ray structure determination (Baskaran and Ang, 2015). The properties of the crystals vary, but they are generally quite large relative to the cell, ranging from 1-2 μm up to hundreds of μm in size (Schönherr et al., 2015). In our recent work, we modified inka-PAK4 crystals to create ftn-PAK4, which is a ferritin-containing crystal that can mineralize enough iron to be attracted to a nearby permanent magnet (Li et al., 2019).The production and isolation of intact protein crystals poses several unique experimental challenges. Because the crystals are so large, purification methods such as gel electrophoresis cannot be used. Likewise, because the crystals are protein-based, harsher lysis conditions such as SDS will disassemble the crystals. However, clean suspensions can be critical for downstream applications. For example, excessive debris in an inka-PAK4 suspension can trap auto-oxidized iron and stick to crystals, generating spurious magnetic attraction results.Here, we present a protocol for the production, isolation, and iron loading of ftn-PAK4 and inka-PAK4 crystals. It is possible to achieve results using only deionized water as the lysis buffer, but we present several optimizations that significantly improve crystal yield and minimize unwanted debris. These considerations should inform future work on other protein crystals, both for their production and isolation as well as their functional applications.
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Acknowledgments
Protocol was based on “Engineering a Genetically Encoded Magnetic Protein Crystal”, in Nano Letters 2019 (Li et al., 2019). This work was supported Stanford Interdisciplinary Graduate Fellowship in association with the Wu Tsai Neurosciences Institute (T.L.L.), Packard Fellowship and Engineering (B.C.), and NIH 1R01GM125737 (B.C.).
Competing interests
The authors declare that there are no competing interests or conflicts of interest.
References
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