Department of Biosystems Science and Engineering, Bioanalytics Group, ETH Zürich, Mattenstrasse 26, Basel, CH-4058, Switzerland.
Department of Biosystems Science and Engineering, Biotechnology and Bioengineering Group, ETH Zürich, Mattenstrasse 26, Basel, CH-4058, Switzerland.
Adv Biosyst. 2020 Nov;4(11):e2000153. doi: 10.1002/adbi.202000153. Epub 2020 Oct 21.
Giant unilamellar lipid vesicles (GUVs) are widely used as model membrane systems and provide an excellent basis to construct artificial cells. To construct more sophisticated artificial cells, proteins-in particular membrane proteins-need to be incorporated in GUVs. However, current methods for protein reconstitution have limited throughput or are not generally applicable for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium-mediated membrane fusion to transfer proteins between negatively charged GUVs and cell-derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Fusion conditions are optimized using large unilamellar vesicles and GUVs containing phosphatidylserines and fusogenic lipids. The approach is then applied to induce lipid mixing and subsequent transfer of the overexpressed membrane receptor from CDVs into GUVs. The membrane receptor is detected by immunofluorescence on GUVs that underwent lipid mixing with CDVs. Those GUVs also exhibit esterase activity because cytosolic esterases entrapped in the CDVs are transferred during membrane fusion. Thus, content mixing is demonstrated. Using CDVs circumvents the need to purify or solubilize proteins. Moreover, calcium-mediated fusion allows transfer of lipids, water-soluble and membrane bound proteins in one step, resulting in a semi-synthetic cell.
巨大单层脂质囊泡（GUVs）被广泛用作模型膜系统，为构建人工细胞提供了极好的基础。为了构建更复杂的人工细胞，需要将蛋白质（特别是膜蛋白）掺入 GUVs 中。然而，当前用于蛋白质重组的方法通量有限，或者不是普遍适用于所有蛋白质，因为它们依赖于去污剂溶解。通过引入钙介导的膜融合来在带负电荷的 GUV 和源自过表达膜受体蛋白的 HEK293T 细胞的质膜囊泡（CDV）之间转移蛋白质，解决了这一限制。使用含有磷脂酰丝氨酸和融合脂质的大单层囊泡和 GUV 优化融合条件。然后将该方法应用于诱导脂质混合以及随后将过表达的膜受体从 CDV 转移到 GUV 中。通过免疫荧光检测与 CDV 发生脂质混合的 GUV 上的膜受体。那些 GUV 还表现出酯酶活性，因为质膜囊泡中的胞质酯酶在膜融合过程中被转移。因此，证明了内容物混合。使用 CDV 避免了纯化或溶解蛋白质的需要。此外，钙介导的融合允许在一步中将脂质、水溶性和膜结合蛋白转移，从而产生半合成细胞。
