Vigentini Ileana, Merico Annamaria, Tutino Maria Luisa, Compagno Concetta, Marino Gennaro
Università degli Studi di Milano, Dipartimento di Scienze Biomolecolari e Biotecnologie, Via Celoria, 26 20133 Milano, Italy.
J Biotechnol. 2006 Dec 15;127(1):141-50. doi: 10.1016/j.jbiotec.2006.05.019. Epub 2006 Jun 12.
The optimization of production strategy is a very useful tool to attain high level of recombinant protein at a low cost. A promising biotechnological application of psychrophilic bacteria is their use as non-conventional host for the recombinant production of useful proteins. The lowering of the expression temperature can in fact facilitate the correct folding of heterologous proteins that accumulate in insoluble form as inclusion bodies when produced in Escherichia coli. An example of such "difficult" proteins is the human nerve growth factor (hNGF). The gene encoding the mature form of hNGF was expressed in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 at 4 degrees C. Western blotting experiments demonstrated that the protein was produced in soluble form and translocated in the periplasmic space. Furthermore, an analytical gel filtration chromatography confirmed that the recombinant protein was largely in dimeric form. For a more efficient recombinant rhNGF production, the influence of cultivation operational strategies and growth conditions (medium composition, temperature, specific growth rate) on biomass yield and recombinant protein production was investigated in batch and chemostat cultivations. The highest product yield of soluble rhNGF (7.5mg(NGF)g(dryweight)(-1)) has been achieved in batch culture at 4 degrees C on Schatz medium with addition of tryptone and vitamins.
生产策略的优化是一种以低成本获得高水平重组蛋白的非常有用的工具。嗜冷菌的一个有前景的生物技术应用是将其用作重组生产有用蛋白质的非常规宿主。事实上，降低表达温度可以促进异源蛋白的正确折叠，这些异源蛋白在大肠杆菌中生产时会以包涵体的形式积累为不溶性形式。这种“难表达”的蛋白质的一个例子是人神经生长因子（hNGF）。编码hNGF成熟形式的基因在南极细菌嗜盐栖假交替单胞菌TAC125中于4℃下表达。蛋白质印迹实验表明该蛋白以可溶形式产生并转运到周质空间。此外，分析凝胶过滤色谱证实重组蛋白主要为二聚体形式。为了更高效地重组生产rhNGF，在分批培养和恒化器培养中研究了培养操作策略和生长条件（培养基组成、温度、比生长速率）对生物量产量和重组蛋白生产的影响。在添加胰蛋白胨和维生素的Schatz培养基上于4℃进行分批培养时，可溶性rhNGF的最高产物产量（7.5mg（NGF）g（干重）-1）得以实现。
