分子克隆、结构建模及重组 Aspergillus terreusl. asparaginase 在大肠杆菌中的生产。
Molecular cloning, structural modeling and production of recombinant Aspergillus terreusl. asparaginase in Escherichia coli.
机构信息
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
出版信息
Int J Biol Macromol. 2018 Jan;106:1041-1051. doi: 10.1016/j.ijbiomac.2017.08.110. Epub 2017 Aug 26.
l-Asparaginase (EC 3.5.1.1) is an important medical enzyme that catalysis the hydrolysis of l-asparagine to aspartic acid and ammonium. For over four decades l. asparaginase utic agent for the treatment of a variety of lymphoproliferative disorders and lymphoma such as acute lymphoblastic leukemia. In the present study A. terreus full length l. asparaginase gene, 1179bp was optimized for expression in Escherichia coli BL21 (DE3) pLysS. The full length A. terreusl. asparaginase gene encoding a protein of 376 amino acids with estimated molecular weight of 42.0kDa and a theoretical isoelectric point (pI) of 5.0. BLAST and phylogeny analysis revealed that the A. terreusl. asparaginase shared high similarity with other known fungal l. asparaginase (75% homology with A. nomius and 71% with A. nidulans). The recombinant protein was overexpressed in the form of amorphous submicron proteinaceous inclusion bodies upon induction with 1mM IPTG at 37°C for 18h.
L-天冬酰胺酶（EC 3.5.1.1）是一种重要的医学酶，可催化 L-天冬酰胺水解为天冬氨酸和铵。四十多年来，L-天冬酰胺酶一直是治疗各种淋巴增生性疾病和淋巴瘤的有效药物，如急性淋巴细胞白血病。本研究对深黄被孢霉全长 L-天冬酰胺酶基因进行优化，用于在大肠杆菌 BL21（DE3）pLysS 中表达。全长 A. terreus L-天冬酰胺酶基因编码一个 376 个氨基酸的蛋白质，估计分子量为 42.0kDa，理论等电点（pI）为 5.0。BLAST 和系统发育分析表明，A. terreus L-天冬酰胺酶与其他已知真菌 L-天冬酰胺酶具有高度相似性（与 A. nomius 同源性为 75%，与 A. nidulans 同源性为 71%）。在 37°C 下用 1mM IPTG 诱导 18 小时后，以无定形亚微米蛋白包涵体的形式过表达重组蛋白。
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