Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger.
Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger.
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DOI：
10.1002/bit.22099
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发表时间：
2009-02-15
影响因子：
3.8
通讯作者：
Shiloach, Joseph
中科院分区：
文献类型：
--
作者：
Chill, Liat;Trinh, Loc;Azadi, Parastoo;Ishihara, Mayumi;Sonon, Roberto;Karnaukhova, Elena;Ophir, Yakir;Golding, Basil;Shiloach, Joseph
人α1蛋白酶抑制剂（α1-PI）在黑曲霉（Aspergillus niger）中进行了克隆和表达。黑曲霉是一种可以在特定培养基中生长并进行糖基化的丝状真菌。以淀粉为碳源，溶解氧浓度为30%，pH 7.0, 28°C，建立了深层培养条件。在40 h左右的时间内，每升活性α1-PI的分泌量为8毫克。研究发现，控制蛋白水解是影响生产的重要因素。考察了不同碳源、pH和温度对蛋白的生成和稳定性的影响，并对产物进行了纯化和表征。重组α1-PI的两个分子量变异体由该真菌产生；这种差异归因于分子的糖基化部分。用PNGAse F处理两种糖蛋白，并通过HPAEC、MALDI/TOF-MS、NSI-MSn和GC-MS对其释放的多糖进行分析。过甲基化n -聚糖的MALDI和NSI全质谱分析表明，两种变体的n -聚糖都含有一系列含有5-20个己糖单位的高甘露糖型聚糖。单糖分析表明，它们由n -乙酰氨基葡萄糖、甘露糖和半乳糖组成。连锁分析表明，半乳糖组分为呋喃烷构象，在末端非还原位置附着。含半乳糖呋喃糖的高甘露糖型n -聚糖是最近发现的由黑曲霉产生的几种糖蛋白的典型结构。
Human alpha one proteinase inhibitor (α1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28°C. Eight milligrams per liter of active α1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant α1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MSn, and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5–20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucosamine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.
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DOI：
10.1515/bchm2.1984.365.2.731
发表时间：
1984-01-01
影响因子：
--
作者：
BEATTY, K;MATHESON, N;TRAVIS, J
通讯作者：
TRAVIS, J
影响因子：
4.1
作者：
BROEKHUIJSEN, MP;MATTERN, IE;VANDENHONDEL, CAMJJ
通讯作者：
VANDENHONDEL, CAMJJ
影响因子：
6.4
作者：
Karnaukhova, Elena;Ophir, Yakir;Shiloach, Joseph
通讯作者：
Shiloach, Joseph
影响因子：
4.1
作者：
KWON, KS;SONG, MY;YU, MH
通讯作者：
YU, MH
影响因子：
3.4
作者：
Kolarich, Daniel;Weber, Alfred;Altmann, Friedrich
通讯作者：
Altmann, Friedrich