Karlsruhe Institute of Technology, Institute for Toxicology and Genetics, Germany.
Biomaterials. 2011 Oct;32(28):6719-28. doi: 10.1016/j.biomaterials.2011.05.069. Epub 2011 Jun 23.
Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O(6)-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.
通过紫外光刻技术制备了图案化的双组分自组装单层膜。一种末端带有二硫键的聚乙二醇（Oligo(ethylene glycol) terminated disulfide）作为惰性基质，可减少非特异性蛋白质吸附和细胞黏附。自组装单层膜（Self-assembled monolayer，SAM）的第二组分呈现苄基鸟嘌呤部分，用于固定融合了突变型 O(6)-烷基鸟嘌呤-DNA 烷基转移酶（SNAP 标签）的 Sonic hedgehog（Shh）。SNAP 标签的酶活性允许连接的 Shh 进行选择性和共价固定。飞行时间二次离子质谱验证了图案化 SAM 中苄基鸟嘌呤头基的正确横向分布。对混合 SAM 中非特异性和特异性蛋白质结合的定量分析表明，随着苄基鸟嘌呤/聚乙二醇（Benzylguanine/(ethylene glycol)）比例的增加，白蛋白的吸附增加。然而，预先吸附的白蛋白不会阻止 SNAP 标记的 Shh 的固定。此外，即使在转染的 HEK293 细胞条件培养基中存在许多竞争蛋白的情况下，所获得的微图案化基底也允许 SNAP 标记的 Shh 的直接固定。因此，所提出的系统适合于从复杂混合物中控制固定融合蛋白，避免了纯化步骤。
