Jevsevar Simona, Gaberc-Porekar Vladka, Fonda Irena, Podobnik Barbara, Grdadolnik Joze, Menart Viktor
Lek Pharmaceuticals d.d., Verovskova 57, SI-1000 Ljubljana, Slovenia.
Biotechnol Prog. 2005 Mar-Apr;21(2):632-9. doi: 10.1021/bp0497839.
Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.
人粒细胞集落刺激因子（hG-CSF）是一种用于肿瘤学的重要生物制药药物，目前主要在大肠杆菌中生产。人hG-CSF基因在大肠杆菌中的表达非常低，因此设计了一种半合成的、密码子优化的hG-CSF基因，并将其亚克隆到pET表达质粒中。这使得产量超过了细胞总蛋白的50%。我们设计了一种低温生物合成的新方法，能够形成“非经典”包涵体，从中可以通过非变性溶剂（如温和洗涤剂或低浓度极性溶剂，如二甲基亚砜和非洗涤剂磺基甜菜碱）轻松提取正确折叠的蛋白质。傅里叶变换红外光谱（FT-IR）分析证实了包涵体相对于生长温度的不同性质，并表明在非经典包涵体中存在大量极有可能正确折叠的还原型hG-CSF。以这种方式获得的正确折叠的功能性hG-CSF产量超过了细胞中产生的总hG-CSF的40%，并且在非变性条件下几乎可以完全提取。在纯化过程中无需包含变性/复性步骤，这使得能够开发出更高效的工艺，其特点是产量更高、成本更低且涉及环境友好型技术。所展示的技术在50升规模上成功运行，生产出质量相同的非经典包涵体。为生产hG-CSF开发的方法可以扩展到其他蛋白质；因此，设想了更广泛的工业开发潜力。
