Suwannaboon Rukkit, Phiwsaiya Kornsunee, Senapin Saengchan, Khunrae Pongsak, Rattanarojpong Triwit
Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Protein Expr Purif. 2013 Dec;92(2):183-9. doi: 10.1016/j.pep.2013.09.012. Epub 2013 Oct 1.
HtrA2 is an apoptosis-activating protein to enhance the apoptotic process by preventing the formation of the IAP-caspase complex, thus freeing caspase to trigger the apoptosis pathway. Here, we presented the full-length sequence of HtrA2 from the black tiger shrimp (PmHtrA2). The full-length PmHtrA2 transcript was 1403 bp with a 1338 bp open reading frame encoding 445 amino acids and contains 5 conserved domains, namely, a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a serine protease domain, and a PDZ domain normally found in HtrA2 proteins of other organisms. The mature form of PmHtrA2 was cloned into the pET28b(+) and pET15bThio vectors, and the expression of the protein was compared in Escherichia coli BL21 DE3 and BL21 RIL (CodonPlus) strains. Greater quantities of stable and soluble PmHtrA2 were expressed as a thioredoxin fusion protein in E. coli BL21 RIL (CodonPlus) cells with the recombinant pET15bThio-PmHtrA2 vector. To investigate the expression of PmHtrA2 in shrimp, the crude proteins from several shrimp tissues were imaged by Western blot using the polyclonal antibody specific to the recombinant PmHtrA2. The expression of the 47-kDa immature PmHtrA2 protein could be detected in shrimp lysates from the gills and the muscles. This study is the first to report the full-length PmHtrA2 gene, which is functional in black tiger shrimp and will lead to more focused studies on the function of PmHtrA2 in apoptosis regulation during immune responses to viral infection in shrimp.
HtrA2是一种凋亡激活蛋白，可通过阻止IAP-半胱天冬酶复合物的形成来增强凋亡过程，从而使半胱天冬酶得以释放以触发凋亡途径。在此，我们展示了来自黑虎虾的HtrA2全长序列（PmHtrA2）。PmHtrA2转录本全长1403 bp，开放阅读框为1338 bp，编码445个氨基酸，包含5个保守结构域，即线粒体靶向信号（MTS）、跨膜（TM）结构域、IAP结合基序（IBM）、丝氨酸蛋白酶结构域和PDZ结构域，这些结构域通常存在于其他生物的HtrA2蛋白中。将PmHtrA2的成熟形式克隆到pET28b(+)和pET15bThio载体中，并在大肠杆菌BL21 DE3和BL21 RIL（CodonPlus）菌株中比较该蛋白的表达。在带有重组pET15bThio-PmHtrA2载体的大肠杆菌BL21 RIL（CodonPlus）细胞中，以硫氧还蛋白融合蛋白的形式表达了大量稳定且可溶的PmHtrA2。为了研究PmHtrA2在虾中的表达情况，使用针对重组PmHtrA2的多克隆抗体通过蛋白质免疫印迹法对几种虾组织的粗蛋白进行成像。在鳃和肌肉的虾裂解物中可检测到47 kDa未成熟PmHtrA2蛋白的表达。本研究首次报道了全长PmHtrA2基因，该基因在黑虎虾中具有功能，这将使对PmHtrA2在虾对病毒感染的免疫反应中凋亡调节功能的研究更加集中。
