A bacterial expression host was used for in vivo incorporation of p-azidophenylalanine (p-N₃Phe), a phenylalanine (Phe) analog, into an artificial extracellular matrix protein (aECM-CS5-ELF). The host harbors the mutant phenylalanyl-tRNA synthetase (PheRS) which has an enlarged binding pocket, where the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) was expressed under the control of T7 promoters. Biosynthesized aECM-CS5-ELF containing p-N₃Phe (aECM-CS5-ELF-N₃) was coupled with alkyne-containing poly(2-oxazoline) prepared via ring-opening polymerization of 2-oxazolines (2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, and 2-iso-propyl-2-oxazoline) using methyl triflate (TfOMe) and propiolic acid as the initiator and terminator, respectively. A copper-catalyzed alkyne-azide click reaction was used to graft the poly(2-oxazoline)s onto the aECM. Examination of the solubility in organic and aqueous media, and the lower critical transition temperature (LCST), revealed a dependence on the incorporation ratio of p-N₃Phe and graft chain species; the LCST behavior was markedly altered when poly(2-oxazoline) moieties were present as side chains. Circular dichroism measurements indicate that grafting was not responsible for conformational changes, because the conformation was retained both at below and above the LCST. Specific adhesion and subsequent temperature-sensitive detachment of human umbilical vein endothelial cells (HUVECs) onto the cross-linked aECM-CS5-ELF-N₃-graft-poly(2-oxazoline) surfaces was also demonstrated.

细菌表达宿主用于体内将苯丙氨酸（Phe）类似物对叠氮苯丙氨酸（p -N ₃ Phe）掺入人工细胞外基质蛋白（aECM-CS5-ELF）。宿主带有突变的苯丙氨酰-t RNA合成酶（PheRS），其具有扩大的结合口袋，其中Ala294Gly / Thr251Gly突变的PheRS（PheRS **）在T7启动子的控制下表达。将生物合成的含p -N ₃ Phe的aECM-CS5-ELF（aECM-CS5-ELF-N ₃）与通过2-恶唑啉（2-甲基-2）的开环聚合制备的含炔的聚（2-恶唑啉）偶联-恶唑啉，2-乙基-2-恶唑啉和2- iso-丙基-2-恶唑啉），分别使用三氟甲磺酸甲酯（TfOMe）和丙酸作为引发剂和终止剂。铜催化的炔-叠氮化物点击反应用于将聚（2-恶唑啉）接枝到aECM上。对有机介质和水性介质的溶解度以及较低的临界转变温度（LCST）进行了研究，结果显示它们依赖于p -N ₃的掺入比苯丙氨酸和接枝链种类；当聚（2-恶唑啉）部分作为侧链存在时，LCST行为显着改变。圆二色性测量表明，嫁接不构成构象变化，因为在LCST的下方和上方均保留了构象。还证明了人脐静脉内皮细胞（HUVEC）在交联的aECM-CS5-ELF-N _3-接枝-聚（2-恶唑啉）表面上的特异性粘附和随后的温度敏感性分离。