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- W4285676500 abstract "involves lysosomal transmembrane proteins whose defect may lead to mild to severe diseases. Due to the difficulty in the study of transmembrane proteins, our current knowledge in transmembrane proteins, especially in organelle transmembrane proteins are still limited. Disrupted in renal carcinoma 2 (DIRC2), has been recently identified as a constituent of lysosomal transmembrane protein. Structurally, the protein belongs to a member of major facilitator superfamily (MFS), a large group of secondary tranporter proteins with diverse substrates and characterized by the presence of, but not limited to, twelve transmembrane spanning domains. DIRC2 shows an unique characteristic that it is fragmented into two nearly equal fragments in the lysosomal fraction of mammalian cells. In vivo study has revealed that cathepsin L is likely to be involved in the proteolysis of DIRC2. In order to analyze the possibility DIRC2 is directly processed by cathepsin L, DIRC2 gene from human needs to be constructed and be expressed in full length within Escherichia coli, which is a comfortable host for heterologous protein expression and has been proven to work well in expressing transporter protein of major facilitator superfamily. Human DIRC2 gene was PCR amplified and ligated with pGEX-2T plasmid. The resulted construct was used to transform E. coli BL21 (DE3) which has an overexpression system for gene placed downstream to its T7 promotor, under induction of isopropyl β-D-1thiogalactopyranoside (IPTG). Expression of DIRC2 in E. coli is discussed and the over-expressed full length DIRC2 provides a prerequisite material to investigate its proteolysis by cathepsin L." @default.
- W4285676500 created "2022-07-17" @default.
- W4285676500 creator A5057092901 @default.
- W4285676500 date "2022-07-17" @default.
- W4285676500 modified "2023-09-25" @default.
- W4285676500 title "Construction and Expression of Human Disrupted in Renal Carcinoma 2 (DIRC2) in Escherichia coli" @default.
- W4285676500 doi "https://doi.org/10.31219/osf.io/x2pvg" @default.
- W4285676500 hasPublicationYear "2022" @default.
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