自组装双亲短肽氨基酸组成及连接肽对其融合酶表达量的影响  

Analysis of the factors influencing the expression of enzymes fused with self-assembling amphipathic peptides

作　　者：赵伟欣 刘松[1,2] 刘立明[3] 陈坚[1,2] 堵国成[1,2] 

机构地区：[1]江南大学,工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]江南大学,食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《食品与发酵工业》2017年第12期1-6,共6页Food and Fermentation Industries

基　　金：国家自然科学基金(31401638);国家自然科学基金(31771913);江苏省重点研发计划社会发展项目(BE2016629)

摘　　要：自组装双亲短肽(self-assembling amphipathic peptides,SAPs)是一类亲疏水氨基酸按一定规律分布,具有自聚合效应的氨基酸短肽,融合在酶蛋白N端时,具有促进表达和稳定化的功能。以自组装双亲短肽S1(AEAEAKAKAEAEAKAK)为出发序列,与来源于Bacillus sp.WSHB04-02的碱性果胶酶(alkaline polygalacturonate lyase,PGL)组成的融合酶为模式蛋白,考察SAPs的氨基酸组成及SAPs融合蛋白内部连接肽(linker peptide)对PGL-SAP融合酶的表达量的影响。结果显示,含有较弱疏水性的甘氨酸和丙氨酸残基的PGL-S1突变体可使融合酶正常表达,其中,含有组氨酸的PGL-S1v1具有相对最高的胞外酶活,较野生型提高了9倍,较PGL-S1提高了1.5倍。连接肽方面,与刚性连接肽相比,含有柔性连接肽的融合酶具有更高的胞外酶活,含有(GGGGS)3的PGL-F-S1融合酶胞外酶活是野生型的14倍。上述结果表明,SAPs的氨基酸组成及融合蛋白之间的连接肽对PGL融合酶的表达具有重要影响。Self-assembling amphipathic peptides（ SAPs） are a kind of functional peptides,which have alternating hydrophilic and hydrophobic residues and can affect the thermal stability and catalytic properties of the fused enzymes. In this study,several SAPs fusion protein comprising S1 peptide（ AEAEAKAKAEAEAKAK） derivatives and Bacillus sp. WSHB04-02 alkaline polygalacturonate lyase（ PGL） or PGL-S1 with different linker peptides were constructed,and the effect of the linker peptides and the amino acids composition of SAPs on expression quantity the fusion enzymes were examined. The results showed that the hydrophobicity of hydrophobic amino acids make a critical contribution on the expression of PGL. The SAPs containing the weaker hydrophobic alanine and glycine residues could lead to normal expression of fusion protein. The PGL-S1 v1 possessed relative high crude enzyme activity. Compared with the PGL and PGL-S1,the extracellular enzyme activity of PGL-S1 v1 was increased by 9-fold and 1. 5-fold,respectively. As for the linker peptides,the flexible linkers efficiently enhanced the extracellular expression of the fusion protein compared with the rigid counterparts. The crude enzyme activity of PGL-F-S1 with flexible linker peptide was increased 14-fold compared with PGL. These results suggested that the amino acids composition of SAPs and the flexibility of the linker peptides could effectively affect the expression quantity the fusion protein.

关 键 词：自组装双亲短肽 融合蛋白 连接肽 氨基酸极性 表达量 碱性果胶酶 

分 类 号：TS201.2[轻工技术与工程—食品科学]