巴西蕉MaCMO和MaBADH原核表达体系筛选及纯化

Prokaryotic expression system screening and purification of MaCMO and MaBADH from Brazilian banana

关键词:  巴西蕉, 胆碱单加氧酶, 甜菜碱醛脱氢酶, 同源克隆, 原核表达

Key words:  Brazilian banana, choline monooxygenase, betaine aldehyde dehydrogenase, homologous cloning, prokaryotic expression

摘要:

胆碱单加氧酶(choline monooxygenase,CMO)和甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)分别是香蕉甘氨酸甜菜碱(glycine betaine,GB)合成过程中的限速酶和关键酶。先前研究在不同基因型香蕉中发现来源于香蕉A、B基因组的CMO和BADH基因存在显著性结构差异,可能导致其功能分化。为了从蛋白水平解析香蕉A、B基因组CMO和BADH酶学特性差异,该研究以巴西蕉(Musa acuminata L. AAA group, cv. Cavendish)为材料,克隆MaCMO和MaBADH编码序列,通过生物信息学分析其结构特征,构建pET28a-MaCMO和pET28a-MaBADH原核表达载体,转化大肠杆菌BL21(DE3),筛选活性蛋白表达最优条件,并利用镍亲和层析柱纯化蛋白,最终确定香蕉CMO和BADH活性蛋白的诱导与纯化方法。结果表明:(1)MaCMO具有加氧酶家族典型的Rieske型 [2Fe-2S]结构域和铁结合位点; MaBADH含有高度保守的醛脱氢酶十肽序列。(2)MaCMO蛋白由425个氨基酸组成,蛋白分子质量47.48 kDa,二级结构以无规则卷曲为主,为亲水性蛋白; MaBADH蛋白由505个氨基酸组成,蛋白分子质量55.10 kDa,二级结构以α-螺旋为主,为亲水性蛋白。(3)BL21(DE3)原核表达系统中,在28 ℃、18 h、IPTG浓度0.5 mmol·L^-1条件下,MaCMO在包涵体中大量表达非活性蛋白; MaBADH活性蛋白在37 ℃、12 h、IPTG浓度0.1 mmol·L^-1时表达量最大,可通过亲和层析法纯化。综上认为,在BL21(DE3)原核表达系统中,MaCMO表达非活性蛋白,MaBADH表达活性蛋白。该研究结果为解析香蕉A、B基因组CMO和BADH的功能差异提供了蛋白水平的理论依据,并为同源基因的功能分化研究提供了技术参考。

Abstract:

Choline monooxygenase(CMO)and betaine aldehyde dehydrogenase(BADH)are the rate-limiting enzyme and key enzyme, respectively, in the glycine betaine(GB)biosynthesis pathway in banana. Previous studies have revealed that CMO and BADH genes derived from the A and B genomes of different banana genotypes exhibit significant structural divergences, which may lead to functional differentiation. To explore the enzymatic differences between CMO and BADH proteins encoded by the A and B genomes at the protein level, this study cloned the coding sequences of MaCMO and MaBADH from Musa acuminata L. AAA group, cv. Cavendish. Bioinformatics analysis was performed to characterize their structural features. The prokaryotic expression vectors pET28a-MaCMO and pET28a-MaBADH were constructed and transformed into Escherichia coli BL21(DE3). Optimal conditions for active protein expression were screened, and nickel affinity chromatography was employed for protein purification. The results were as follows:(1)MaCMO contained the Rieske-type [2Fe-2S] domain and iron-binding sites typical of oxygenase family proteins, while MaBADH harbors a highly conserved decapeptide motif of aldehyde dehydrogenases.(2)MaCMO consisted of 425 amino acids with a molecular mass of 47.48 kDa, exhibiting a predominantly random coil secondary structure and hydrophilic properties; MaBADH comprised 505 amino acids(55.10 kDa), characterized by an α-helix-rich secondary structure and hydrophilicity.(3)In the BL21(DE3)prokaryotic expression system, MaCMO formed inactive inclusion bodies under induction at 28 ℃ for 18 h with 0.5 mmol·L^-1 IPTG, whereas MaBADH achieved maximal expression of active protein at 37 ℃ for 12 h with 0.1 mmol·L^-1 IPTG and could be efficiently purified via affinity chromatography. In conclusion, the BL21(DE3)system expresses MaCMO as inactive aggregates but produces functional MaBADH. This study provides a protein-level theoretical basis for elucidating functional divergence between CMO and BADH encoded by the A and B genomes in bananas and offers methodological insights for comparative functional studies of homologous genes across plant genomes.