Dolinska Monika B, Wingfield Paul T, Sergeev Yuri V
National Eye Institute, National Institutes of Health, Bethesda, Maryland.
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland.
Curr Protoc Protein Sci. 2017 Aug 1;89:6.15.1-6.15.12. doi: 10.1002/cpps.37.
The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography. The purified recombinant human tyrosinase is a soluble monomeric glycoprotein with an activity that mirrors the tyrosinase in vivo function. © 2017 by John Wiley & Sons, Inc.
本文描述了从感染杆状病毒载体的昆虫幼虫中纯化一种酶的过程。酪氨酸酶这种酶具有生物医学重要性，它催化黑色素生成过程中的首个限速步骤。酪氨酸酶突变可导致1型眼皮肤白化病（OCA1），这是一种遗传性眼病，与黑色素生成减少和视力缺陷有关。为了简化表达及后续纯化，将细胞外结构域在昆虫细胞中表达，在粉纹夜蛾幼虫中生产，并通过亲和色谱和尺寸排阻色谱进行纯化。纯化后的重组人酪氨酸酶是一种可溶性单体糖蛋白，其活性反映了酪氨酸酶在体内的功能。© 2017约翰威立国际出版公司。
