六聚组氨酸标记的人组织转谷氨酰胺酶在大肠杆菌中的表达及纯化

Expression in Escherichia coli and purification of hexahistidine-tagged human tissue transglutaminase.

作者信息

Shi Qingli, Kim Soo-Youl, Blass John P, Cooper Arthur J L

机构信息

Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10021, USA.

出版信息

Protein Expr Purif. 2002 Apr;24(3):366-73. doi: 10.1006/prep.2001.1587.

DOI:10.1006/prep.2001.1587

PMID:11922752

Abstract

Recent evidence suggests that aberrant transglutaminase activity is associated with a wide variety of diseases. Tissue transglutaminase is the most widely distributed of the six well-characterized transglutaminases in humans. We describe a method for expressing hexahistidine-tagged human tissue transglutaminase in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with CelLytic B Bacterial Cell Lysis/Extraction Reagent was accomplished by immobilized metal (Ni2+) affinity column chromatography. The procedure typically yields highly purified and highly active recombinant human tissue transglutaminase in about 1 day (about 0.6 mg/from a 1-liter culture).

摘要

最近的证据表明，异常的转谷氨酰胺酶活性与多种疾病相关。组织转谷氨酰胺酶是人类六种特征明确的转谷氨酰胺酶中分布最广泛的一种。我们描述了一种使用pET-30 Ek/LIC表达载体在大肠杆菌BL21(DE3)中表达六聚组氨酸标记的人组织转谷氨酰胺酶的方法。通过固定化金属（Ni2+）亲和柱色谱法从用CelLytic B细菌细胞裂解/提取试剂处理的大肠杆菌细胞悬浮液中纯化表达的酶。该方法通常在约1天内（从1升培养物中约0.6毫克）产生高度纯化且高活性的重组人组织转谷氨酰胺酶。