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3 Q&A 3360 Views Jul 20, 2020
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.
0 Q&A 3571 Views Jul 5, 2019
In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on laser-triggered Giant Unilamellar Vesicles (GUVs) fusion. Our protocol allows sequential addition of proteins to a negatively curved membrane, while at the same time controlling the buffer composition, lipid composition and membrane tension. Moreover, this method does not require a step of protein detachment, greatly simplifying the process of protein encapsulation over existing methods.

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