Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens
Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens
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DOI：
10.1016/j.pep.2011.03.002
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发表时间：
2011-07-01
影响因子：
1.6
通讯作者：
Retallack, Diane M.
中科院分区：
文献类型：
--
作者：
Jin, Hongfan;Cantin, Greg T.;Retallack, Diane M.
在细菌系统中成本有效地生产可溶性重组蛋白在所表达的靶蛋白的表达水平和质量方面仍然存在问题。这些限制在今天具有特殊的意义，因为创新蛋白质药物的“生物类似药”版本正在进入临床和市场。采用高通量、平行处理的表达菌株工程方法，研究了人粒细胞集落刺激因子（G-CSF）在荧光假单胞菌中的可溶性表达。优化人g-csf基因在荧光假单胞菌中的表达，并克隆到一组周质表达载体中。将这些质粒转化到各种荧光假单胞菌宿主菌株中，每种宿主菌株具有独特的表型，以评价96孔生长和蛋白表达形式中的可溶性表达。为了鉴定产生高水平的完整的可溶性Met-G-CSF产物的菌株，使用生物层干涉测量法（BLI）平行筛选来自Pfenex Expression Technology（TM）工具箱的150多种蛋白酶缺陷宿主菌株，以定量活性G-CSF与其受体的结合。通过LC-MS分析筛选这些菌株的子集，以评估表达的G-CSF蛋白的质量。鉴定了在pfal基因中具有抗生素抗性标记插入的单一菌株，其产生> 99%的Met-GCSF。构建了从基因组中完全缺失自身转运蛋白编码基因pfal的宿主，并且观察到在1升发酵规模下该菌株中可溶性活性Met-GSCF的表达为350 mg/L。(C)2011 Elsevier Inc. All rights reserved.
Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfenex Expression Technology (TM) toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfal gene was identified that produced > 99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfal from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350 mg/L at the 1 liter fermentation scale. (C) 2011 Elsevier Inc. All rights reserved.