小鼠β-神经生长因子蛋白的互补DNA（cDNA）在大肠杆菌中的表达

Expression of the cDNA for mouse beta-nerve growth factor protein in Escherichia coli.

作者信息

Hu G L, Neet K E

机构信息

Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106.

出版信息

Gene. 1988 Oct 15;70(1):57-65. doi: 10.1016/0378-1119(88)90104-7.

DOI:10.1016/0378-1119(88)90104-7

PMID:3071493

Abstract

The cDNA coding for the mature beta-nerve growth factor (beta-NGF) has been cloned into a plasmid expression vector, pAS1, and expressed in Escherichia coli. The cDNA fragment in pAS1 is under the control of strong phage transcriptional and translational initiation elements that provide for regulated expression of cloned genes in E. coli. The protein, produced in bacteria at a level of about 0.0005-0.1% of cell protein, was purified by ammonium sulfate precipitation and ion exchange chromatography. The recombinant NGF was biologically active in the PC12 neurite outgrowth assay, and formed a band at Mr of about 11,000 to 12,000, when electrophoresed on sodium dodecyl sulfate-polyacrylamide gel and Western-blotted.

摘要

编码成熟β-神经生长因子（β-NGF）的cDNA已被克隆到质粒表达载体pAS1中，并在大肠杆菌中表达。pAS1中的cDNA片段受强噬菌体转录和翻译起始元件的控制，这些元件可调控克隆基因在大肠杆菌中的表达。该蛋白在细菌中产生的水平约为细胞蛋白的0.0005-0.1%，通过硫酸铵沉淀和离子交换色谱法进行纯化。重组NGF在PC12神经突生长试验中具有生物活性，在十二烷基硫酸钠-聚丙烯酰胺凝胶上电泳并进行蛋白质印迹时，在约11,000至12,000的分子量处形成一条带。