Berzins V, Jansone I, Skangals A, Kalnins P, Liepa S, Baumanis V
Institute of Molecular Biology, University of Lativia, Riga.
J Biotechnol. 1993 Aug;30(2):231-43. doi: 10.1016/0168-1656(93)90116-5.
A synthetic DNA sequence coding for the 28 amino acid residues of alpha-human atrial natriuretic factor (alpha-hANF) and the N-terminal linker tripeptide Ile-Asp-Lys was inserted in the 3'-terminal part of the RNA bacteriophage fr coat protein gene. The cloned hybrid gene was isolated and placed into an expression vector under the control of the inducible E. coli tryptophan promoter and phage fr coat protein translation initiation region (TIR) sequence. In an appropriate host strain the expressed fusion protein accounts for at least 10% of the total cellular protein. In order to achieve high-cell density in a bioreactor while maintaining efficiency of alpha-hANF expression, improved cultivation conditions were selected using modified Shielach-Bauer's culture media containing glucose, yeast extract and bacto tryptone at an initial concentration of 2 g l-1 of each, adding concentrate of medium throughout the microbial growth and maintaining the dissolved oxygen in a range of 25-30%. At 13-14 h cultivation, the cell density reached 40 g cell dry weight per liter and the yield of fusion protein exceeded 45 mg g-1 cell dry weight. Fusion protein from solubilized E. coli cells was purified to homogeneity by ion exchange chromatography on DEAE-, CM-cellulose, QAE Sephadex A25 columns and selective precipitation.
编码α-人心房钠尿肽（α-hANF）28个氨基酸残基以及N端连接三肽异亮氨酸-天冬氨酸-赖氨酸的合成DNA序列，被插入到RNA噬菌体fr外壳蛋白基因的3'末端部分。克隆的杂交基因被分离出来，并置于一个受诱导型大肠杆菌色氨酸启动子和噬菌体fr外壳蛋白翻译起始区域（TIR）序列控制的表达载体中。在合适的宿主菌株中，表达的融合蛋白占细胞总蛋白的至少10%。为了在生物反应器中实现高细胞密度，同时保持α-hANF的表达效率，选择了改良的培养条件，使用含有葡萄糖、酵母提取物和细菌胰蛋白胨的改良希拉赫-鲍尔培养基，每种成分的初始浓度为2 g l-1，在微生物生长过程中添加培养基浓缩液，并将溶解氧维持在25%-30%的范围内。在培养13-14小时时，细胞密度达到每升40 g细胞干重，融合蛋白产量超过45 mg g-1细胞干重。通过在DEAE-、CM-纤维素、QAE Sephadex A25柱上进行离子交换色谱和选择性沉淀，将溶解的大肠杆菌细胞中的融合蛋白纯化至同质。
