High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli
High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli
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DOI：
10.1016/j.matbio.2004.11.004
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发表时间：
2005-02-01
期刊：
影响因子：
6.9
通讯作者：
Myllyharju, J
中科院分区：
文献类型：
--
作者：
Neubauer, A;Neubauer, P;Myllyharju, J
胶原蛋白脯氨酰4-羟化酶（C-P4 H），驻留在内质网的内腔内的酶，在所有胶原蛋白的合成中起核心作用。脊椎动物酶是α（2）β（2）四聚体，其中两个催化位点位于α亚基中，蛋白质二硫键异构酶作为β亚基。在体外无细胞系统中从其亚基组装活性C-P4 H四聚体的所有尝试都是不成功的，但是已经报道了在几种细胞类型中通过共表达两种类型的亚基组装重组酶。本文通过在大肠杆菌菌株BL 21和RB 791中的周质表达获得活性I型C-P4 H四聚体。通过在硫氧还蛋白还原酶和谷胱甘肽还原酶突变体大肠杆菌菌株的细胞质中逐步调节其亚基的共表达来进一步优化生产，导致大量的人I型C-P4 H四聚体。从细胞质表达中纯化的C-P4 H四聚体的比活性在作为非重组酶分离的或在昆虫细胞内质网中产生的人I型C-P4 H的报道值范围内，但在发酵罐中的表达水平约为25 mg/l，是在昆虫细胞中获得的表达水平的约5-10倍。在E.大肠杆菌中的蛋白质不同于体内存在的蛋白质和其它宿主中产生的蛋白质，因为它缺乏α亚基的N糖基化，这在结晶实验中可能是有利的。(c)2004 Elsevier B.V/国际基质生物学学会。All rights reserved.
The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments. (c) 2004 Elsevier B.V/International Society of Matrix Biology. All rights reserved.