Wilkinson D L, Ma N T, Haught C, Harrison R G
School of Chemical Engineering and Materials Science, University of Oklahoma, Norman 73019-0628, USA.
Biotechnol Prog. 1995 May-Jun;11(3):265-9. doi: 10.1021/bp00033a004.
A fusion protein that contains human atrial natriuretic peptide (ANP) at its carboxy terminus has been genetically engineered with the objective of being able to produce the peptide in a process with a relatively simple purification procedure. The fusion protein also includes a (His)6 metal affinity binding site at the amino terminus, followed by Escherichia coli thioredoxin, a factor Xa protease recognition site, and ANP. With induction of the tac promoter at 30 degrees C, the expression level of the fusion protein was high (10% of total cell protein as measured by densitometry) and it was almost completely (92%) expressed as a soluble protein in the cytoplasm. A step gradient elution with imidazole of a column of Ni2+ chelated to iminodiacetic acid-agarose saturated with proteins in crude cell extract gave a very nearly pure fusion protein. After digestion of the purified fusion protein with factor Xa protease, ANP of exactly the correct size (to within 2 Da) was observed by coupled HPLC/mass spectrometry.
一种在其羧基末端含有人心房利钠肽（ANP）的融合蛋白已通过基因工程构建，目的是能够在一个纯化过程相对简单的工艺中生产该肽。该融合蛋白在氨基末端还包括一个（His）6金属亲和结合位点，接着是大肠杆菌硫氧还蛋白、一个凝血因子Xa蛋白酶识别位点和ANP。在30℃诱导tac启动子，融合蛋白的表达水平很高（通过光密度法测定占总细胞蛋白的10%），并且几乎完全（92%）以可溶性蛋白的形式表达于细胞质中。用咪唑对镍离子螯合到亚氨基二乙酸 - 琼脂糖上且已被粗细胞提取物中的蛋白质饱和的柱子进行分步梯度洗脱，得到了几乎纯的融合蛋白。在用凝血因子Xa蛋白酶消化纯化的融合蛋白后，通过联用高效液相色谱/质谱法观察到了大小完全正确（误差在2道尔顿以内）的ANP。
