通过与人血清白蛋白融合增强毕赤酵母中粒细胞集落刺激因子的表达

Moolchand Sigar, N. Maity, S. Mishra
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引用次数: 11

摘要

摘要蛋白融合技术已成为提高异种表达系统中治疗蛋白表达水平和半衰期的重要策略之一。粒细胞集落刺激因子(G-CSF)是一种造血生长因子,临床上用于治疗中性粒细胞减少症。通过构建与人血清白蛋白(HSA)的融合蛋白,在毕赤酵母中实现了G-CSF的增强表达和稳定性。该策略包括聚合酶链反应(PCR)扩增密码子优化的G-CSF和HSA结构域3对应的片段。采用重叠PCR技术获得了全长1184 bp的融合基因,连接序列为15 bp,包含4个Gly残基和1个Ser残基。在醇氧化酶1启动子调控下,通过ppicz - α b在α-因子分泌信号序列下游进行细胞外表达。在转化子中观察到110-380 mg L−1的蛋白分泌。研究了曝气和温度对其中一个转化子(35)的影响,融合蛋白过表达和G-CSF水平分别提高了1.8倍和2.3倍。生物活性分析表明,融合蛋白保持了与商用G-CSF制剂相似的细胞增殖活性。
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Enhancing granulocyte colony-stimulating factor expression in Pichia pastoris through fusion with human serum albumin
ABSTRACT Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110–380 mg L−1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.
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