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首页> 外文期刊>Protein Expression and Purification >Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V)
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Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V)

机译：纯化和表征在大肠杆菌中表达的重组抗血管生成的kringle片段：人载脂蛋白（a）（kringle IV（9）-kringle IV（10）-kringle V）的三-kringle片段的纯化和表征

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摘要

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetomtrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF. (C) 2005 Elsevier Inc. All rights reserved.
机译：来自人载脂蛋白（a）（称为LK68）的kringle片段（IV（9）-IV（10）-V型）在大肠杆菌的包涵体中表达。使该包涵体中的LK68溶于尿素，并在存在re-dox对的情况下通过氧化有效地重折叠。然后通过两步离子交换色谱法纯化重折叠的LK68，通过制备型反相色谱法浓缩，然后冷冻干燥，最终收率约为30％。纯化的LK68对赖氨酸和纤维蛋白原表现出深厚的亲和力，这表明该蛋白可以适当折叠，并且还表明了载脂蛋白（a）的天然特性得以保留。在20％（v / v）乙腈存在下，通过反相HPLC分析和尺寸排阻HPLC分析，确定纯化的LK68具有高度均相性。但是，在不使用乙腈的体积排阻HPLC分析中，确定其有些不均一，这通过包括PAGE和IEF在内的天然分析得到了证实。 （C）2005 Elsevier Inc.保留所有权利。

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