Mistry A R, Falciola L, Monaco L, Tagliabue R, Acerbis G, Knight A, Harbottle R P, Soria M, Bianchi M E, Coutelle C, Hart S L
Imperial College School of Medicine at St. Mary's, London, England, UK.
Biotechniques. 1997 Apr;22(4):718-29. doi: 10.2144/97224rr01.
This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.
本文描述了基于甲基营养型酵母毕赤酵母表达系统生产重组蛋白的过程。利用该系统高效生产了大鼠高迁移率族蛋白1（HMG1）。两种形式的HMG1被分泌到培养基中：一种24.5 kDa的物种对应天然HMG1，另一种32 kDa的糖基化衍生物。非糖基化重组HMG1易于纯化，并显示出与从小牛胸腺中纯化的HMG1具有相同的DNA结合特性。与重组HMG1复合的质粒DNA被培养中的多种哺乳动物细胞摄取。观察到荧光素酶报告基因的瞬时表达。在选择性条件下，由于整合到基因组中，新霉素基因得以稳定表达。HMG1介导的基因传递与磷酸钙介导的转染效率相同，但不会造成相关细胞损伤。此外，与磷酸钙法获得的数量高度不可预测相比，在选择G418抗性后获得的稳定转染子通常仅整合一份转染DNA。HMG1转染复合物即使在高浓度下对培养细胞也无毒。
