Maity Nitu, Thawani Ankita, Sharma Anshul, Gautam Ashwani, Mishra Saroj, Sahai Vikram
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
Lilly Hall of Biological Sciences, Purdue University - West Lafayette, Indiana, USA.
Appl Biochem Biotechnol. 2016 Jan;178(1):159-72. doi: 10.1007/s12010-015-1865-y. Epub 2015 Sep 26.
Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.
粒细胞集落刺激因子（GCSF）因其在不同类型的中性粒细胞减少症和化疗引起的白细胞减少症中已证实的疗效而具有治疗应用价值。从NCBI获取的原始564 bp核苷酸序列经过密码子优化，并通过重叠PCR方法进行组装，该方法由16条长度为50 nt且具有15个碱基重叠的寡核苷酸组成。合成基因（CO-GCSF）被克隆到利用甘油醛3-磷酸脱氢酶（GAP）的葡萄糖利用型和利用甲醇的醇氧化酶（AOX1）启动子下，并在毕赤酵母SMD1168菌株中表达。在GAP启动子下的组成型表达导致细胞毒性，而AOX1启动子控制的表达则很稳定。在转化菌落中观察到表达水平的差异，转化子#2分泌的GCSF高达约4 mg/L。在毕赤酵母中表达的GCSF的分子量约为19.0 kDa。通过高度可重复的凝胶光密度法对表达的蛋白质进行定量。研究了几种操作和营养条件对GCSF生产的影响，结果表明有一种通用方法可使GCSF产量比目前采用的标准条件提高几倍（2至5倍）。在5 L发酵罐中的化学限定培养基中培养单拷贝整合体，在诱导期结束时的体积生产率约为0.7 mg/L/h，这比摇瓶培养获得的生产率高约4倍。
