野生大豆肌醇磷酸水解酶Gs5PTase8的原核表达纯化及活性鉴定

Prokaryotic expression, purification, and activity of the inositol polyphosphate 5-phosphatase Gs5PTase8 from wild soybean

摘要:

多磷酸肌醇-5-磷酸酶(inositol polyphosphate 5-phosphatase,5PTase)是肌醇信号传导途径中的关键酶，能水解肌醇磷酸(inositol phosphate,IP)或磷脂酰肌醇磷酸(phosphatidylinositol phosphates,PIP)的肌醇环5-磷酸。然而，大豆中该类基因研究较少。本研究从野生大豆(Glycine soja S.&Z.)中克隆出耐盐基因Gs5PTase8并对其底物进行探究。Gs5PTase8编码493个氨基酸。经序列比对与系统进化树分析，发现其在植物中具有保守性。利用实时荧光定量PCR对不同大豆组织分析该基因的表达，发现Gs5PTase8主要在大豆根中表达。为了探究其水解底物，分别构建了大肠杆菌表达载体pET28a-Gs5PTase8和pGEX4T1-Gs5PTase8，但只成功诱导表达了GST-Gs5PTase8重组蛋白。使用不同浓度异丙基β-d-硫代半乳糖苷(isopropyl beta-d-thiogalactopyranoside,IPTG)，分别在16、30、37℃条件下进行诱导表达条件优化，发现重组蛋白在16℃、0.2 mmol/L IPTG过夜诱导时表达量最高。SDS-PAGE检测重组蛋白的相对分子量约为75 kDa，经纯化后条带单一，纯度达95%以上，二辛可酸法(bicinchoninic acid assay,BCA)检测出重组蛋白的得率为4.9 mg/L。体外底物酶活检测表明，Gs5PTase8蛋白可以水解IP₃[inositol (1,4,5) trisphosphate]、IP₄[inositol (1,3,4,5) tetrakisphosphate]、PI (4,5) P₂[phosphatidylinositol (4,5) bisphosphate]和PI (3,4,5) P₃[phosphatidylinositol (3,4,5) trisphosphate]，本研究为进一步探索Gs5PTase8参与耐盐的分子作用机制提供了科学依据。

Abstract:

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP₃ [inositol(1,4,5)trisphosphate], IP₄ [inositol(1,3,4,5)tetrakisphosphate], PI(4,5)P₂ [phosphatidylinositol(4,5) bisphosphate] and PI(3,4,5)P₃ [phosphatidylinositol(3,4,5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.