Li Thomas L, Cui Bianxiao
Department of Chemistry, Stanford University, Stanford, California, USA.
Bio Protoc. 2020 Jul 20;10(14):e3684. doi: 10.21769/BioProtoc.3684.
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.
蛋白质工程的进展使得在活细胞内生产自组装蛋白质晶体成为可能。我们最近发表的文章展示了ftn-PAK4的生产，ftn-PAK4是一种含铁蛋白的晶体，在分离时能够使铁矿化并具有磁性。我们已经开发出一种优化的方案来生产和分离基于PAK4的晶体。这些晶体首先在传代次数较少的HEK293T细胞中生长，使用含有NP-40和脱氧核糖核酸酶的裂解缓冲液释放，然后在仔细的离心条件下收集。我们的方案使晶体的纯度和产量最大化，而且快速又简便。
