Production, purification and characterization of recombinant human interferon γ
Production, purification and characterization of recombinant human interferon γ
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DOI：
10.1016/0021-9673(92)85539-6
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发表时间：
1992-06
影响因子：
4.1
通讯作者：
Z. Zhang;K. Tong;M. Belew;T. Pettersson;J. Janson
中科院分区：
文献类型：
--
作者：
Z. Zhang;K. Tong;M. Belew;T. Pettersson;J. Janson
基本上三步色谱纯化程序，即，采用离子交换层析、固定化金属离子亲和层析和分子排阻层析等方法，从大肠杆菌基因工程菌产生的包涵体中纯化重组人γ-干扰素（rhIFN-γ）。复性rhIFN-γ的浑浊溶液的分批吸附避免了高速离心以澄清悬浮液的需要。该步骤有效去除了约70%的外源蛋白质杂质。所建立的纯化工艺具有重现性，总回收率为32%。中试加工E.在30-1发酵罐中生长的大肠杆菌细胞产生约70 mg的rhIFN-γ的均质制剂。纯化后的rhIFN-γ的比生物学活性为100%。3.4· 107I.U./毫克蛋白质，这是可比的，其天然对应物。它是碱性蛋白（pI> pH 9），单体相对分子质量为15000。然而，其在尺寸排阻色谱上表现为二聚体。其部分NH 2-末端序列与rhIFN-γ中确定的序列相同。然而，其氨基酸组成和相对分子质量（15 067，通过电喷雾质谱法测定）表明，纯化的蛋白质是一个截短的形式，缺乏15个氨基酸残基从其羧基末端侧。这种修饰似乎对其生物学效力没有任何不利影响。测定了最终产品中的DNA、细菌内毒素和Ni（II）离子的水平。
An essentially three-step chromatographic purification procedure,i.e., ion-exchange, immobilized metal ion affinity and size-exclusion chromatography, is described for the purification to homogeneity of recombinant human interferon-γ (rhIFN-γ) from the inclusion bodies produced in genetically transformedEscherichia colicells. Batchwise adsorption of the cloudy solution of renatured rhIFN-γ obviated the need for high-speed centrifugation to clarify the suspension. This step effectively removed about 70% of extraneous protein impurities. The established purification process is reproducible and leads to a total recovery of 32%. Pilot-scale processing ofE. colicells grown in a 30-1 fermentor gave about 70 mg of a homogeneous preparation of rhIFN-γ. The specific biological activity of purified rhIFN-γ isca. 3.4 · 107I.U./mg protein, which is comparable to that of its natural counterpart. It is basic protein (pI> pH 9) with a monomer relative molecular mass of 15 000. It behaves, however, as a dimer on size-exclusion chromatography. Its partial NH2-terminal sequence is identical with that established for the rhIFN-γ. However, its amino acid composition and its relative molecular mass (15 067 as determined by electrospray mass spectrometry) indicate that the purified protein is a truncated form lacking fifteen amino acid residues from its carboxyl-terminal side. This modification does not seem to have any adverse effect on its biological potency. The levels of DNA, bacterial endotoxins and Ni(II) ions in the final product were determined.