Balkhi Sayed Sharif, Hojati Zohreh
Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, 81746-73441 Iran.
Indian J Clin Biochem. 2021 Apr;36(2):159-166. doi: 10.1007/s12291-020-00872-y. Epub 2020 Jan 30.
Intervening proteins (Inteins) are identified as protein domains in a precursor protein structure. Inteins can excise itself from precursor protein and join the remaining portions which result in forming an active protein. In this study, the transcript expression level of recombinant human Interferon beta (rhIFNβ) connected to the self-cleavage Intein-ELK16 (LELELKLKLELELKLK) tag was measured by real-time PCR in HEK293T cell line. First, the sequence of RecA (Mtu recA) was obtained from the InBase database to do appropriate changes including adding the restriction sites, kozak sequence, signal peptide and ELK16 sequence by SnapGene software. The RNA secondary structure were also examined using the online RNA Fold 2.2 web server. Next, the construct was inserted into pUC19 plasmid. The sequence of rhIFNβ was also cloned into pBudCE4.1 vector. In the next step, the rhIFNβ was ligated into the construct (self-cleavage tag of ELK16) using T4 DNA ligase and the recombinant construct was transfected into HEK293T cell line. Finally, expression of the cassette was evaluated by real-time PCR. The analysis of secondary RNA structure indicates a minimum free energy of MEF - 261.10 kcal/mol. Our results indicate that IFNβ was upregulated (37.8-fold, < 0.0001) in cells which transfected by rhIFNβ-ELK16 compared to the mock and un-transfected conditions. Altogether, our results show that the presence of mini self-cleavage Intein-ELK16 tag along with the rhIFNβ had no interference in transcription of rhIFNβ in the HEK293T cell line.
内含肽被鉴定为前体蛋白结构中的蛋白质结构域。内含肽能够从前体蛋白中自我切割出来，并将剩余部分连接起来，从而形成一个活性蛋白。在本研究中，通过实时PCR在HEK293T细胞系中测量了与自我切割内含肽ELK16（LELELKLKLELELKLK）标签相连的重组人干扰素β（rhIFNβ）的转录表达水平。首先，从InBase数据库中获取RecA（Mtu recA）序列，使用SnapGene软件进行适当修改，包括添加限制性酶切位点、kozak序列、信号肽和ELK16序列。还使用在线RNA Fold 2.2网络服务器检查RNA二级结构。接下来，将构建体插入pUC19质粒。rhIFNβ的序列也被克隆到pBudCE4.1载体中。在下一步中，使用T4 DNA连接酶将rhIFNβ连接到构建体（ELK16的自我切割标签）中，并将重组构建体转染到HEK293T细胞系中。最后，通过实时PCR评估盒式结构的表达。RNA二级结构分析表明最小自由能为MEF - 261.10千卡/摩尔。我们的结果表明，与模拟和未转染条件相比，rhIFNβ-ELK16转染的细胞中IFNβ上调（37.8倍，<0.0001）。总之，我们的结果表明，微型自我切割内含肽ELK16标签与rhIFNβ的存在对HEK293T细胞系中rhIFNβ的转录没有干扰。
