Babaeipour Valiollah, Khanchezar Sirwan, Mofid Mohammad Reza, Pesaran Hagi Abbas Mahdi
Dept. of Bioscience and Biotechnology, Malek Ashtar University of Technology, P.O. Box 14395-1561, Tehran, Iran.
Dept. of Biotechnology, Chemical Engineering Faculty, Tarbiat Modarres University, Tehran, Iran.
Iran Biomed J. 2015;19(2):102-10. doi: 10.6091/ibj.1338.2015.
The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients.
An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h⁻¹ at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography.
Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l⁻¹ and 32.1 g.l⁻¹. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g⁻¹ DCW and 1.83 g.l⁻¹.h⁻¹, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen and PD-Grastim showed that all proteins have a similar secondary structure.
Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity.
蛋白质激素粒细胞集落刺激因子（GCSF）可刺激白细胞生成，在癌症患者的医学治疗中发挥重要作用。
开发了一种在大肠杆菌BL21（DE3）中异源表达人GCSF的高效方法。调整补料速率以在临界值下实现最大可达到的比生长速率。在此方法中，比生长速率在补料开始时保持在最大值0.55 h⁻¹，在诱导时保持在0.4 h⁻¹。重组人GCSF（rh-GCSF）以包涵体形式产生。首先，通过细胞破碎释放包涵体，然后进行洗涤、溶解和复性。最后，通过阳离子交换色谱法纯化rh-GCSF。
显然，较高的比生长速率可缩短工艺时间，从而提高生产率。生物量和GCSF的最终浓度分别达到126 g DCW·L⁻¹和32.1 g·L⁻¹。此外，rh-GCSF的最终比产率（YP/X）和总生产率分别为254 mg·g⁻¹ DCW和
