cDNA cloning and bacterial expression of the human type I keratin 16.
cDNA cloning and bacterial expression of the human type I keratin 16.
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人 I 型角蛋白 16 的 cDNA 克隆和细菌表达。
DOI：
10.1006/bbrc.1995.2495
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发表时间：
1995
影响因子：
3.1
通讯作者：
P. Coulombe
中科院分区：
文献类型：
--
作者：
R. Paladini;K. Takahashi;T. Gant;P. Coulombe
人I型角蛋白16在许多复杂的上皮组织（包括皮肤）中组成型表达，但更为人所知的是其在有利于增强增殖或异常分化的条件下的诱导，包括伤口愈合、牛皮癣和癌症。我们克隆了编码序列的人K16通过应用一个耦合的逆转录-聚合酶链反应程序从培养的人皮肤角质形成细胞制备的mRNA。然后，我们在E.大肠杆菌中，并通过阴离子交换层析纯化溶解的蛋白。回收的重组蛋白在阴离子交换剂上的行为类似于人K14（一种相关的酸性角蛋白），在SDS-PAGE（M（r）48 kD）上与天然人K16共迁移，并与针对人K16的抗血清反应。根据获得的核苷酸序列和相应的重组蛋白的性质，我们得出结论，我们已经克隆了人K16 cDNA的编码部分。在这项研究中获得的序列数据进行比较，早期的报告，人类K16序列，这是在许多方面相互矛盾。纯化重组形式的K16的可用性将使我们能够研究其特性如何与其在伤口愈合和皮肤疾病中的功能相关。
The human type I keratin 16 is constitutively expressed in a number of complex epithelial tissues, including skin, but is better known for its induction under conditions favoring enhanced proliferation or abnormal differentiation, including wound healing, psoriasis, and cancer. We cloned the coding sequence of human K16 by applying a coupled reverse transcription-polymerase chain reaction procedure to mRNAs prepared from cultured human skin keratinocytes. We then expressed the human K16 coding sequence in E. coli and purified the solubilized protein by anion-exchange chromatography. The recombinant protein recovered behaves similarly to human K14 (a related acidic keratin) on the anion-exchanger, co-migrates with native human K16 on SDS-PAGE (M(r) 48 kD), and reacts with antisera directed against human K16. Based on the nucleotide sequence obtained and the properties of the corresponding recombinant protein, we conclude that we have cloned the coding portion of the human K16 cDNA. The sequence data obtained in this study is compared to earlier reports of the human K16 sequence, which are conflicting in many respects. The availability of K16 in a purified recombinant form will allow us to study how its properties may relate to its function during wound healing and in skin diseases.