Protein Expression, Purification, and Structural Characterization of Truncated Human TREM-Like Transcript-2

• Main Authors: Jian-Sheng Chang, 張建盛
• Other Authors: Shou-Lin Chang
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/39999279592359434152

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 95 === TREMs, triggering receptor expressed on myeloid cells, belong to a rapidly expanding family of receptors that include activating and inhibitory isoforms encoded by a gene cluster linked to the major histocompatibility complex (MHC) on human chromosome 6. Human TREM-like transcript-2 (hTLT-2), one kind of TREM gene family with 321 amino acids, is a membrane protein and signal receptor expressed on neutrophils, macrophages, and B lymphocytes in innate and adaptive immune system. Until now, the characterization and structural analysis of hTLT-2 remain unclear. In this study, the N-terminus extracellular domain of hTLT-2 was truncated to 26-kDa (from 14-Q to 265-D) and 13-kDa (from 14-Q to 132-N) fragments and cloned into expression vectors pET-28a and pGEX-6P-3. The two partial fragments of hTLT-2 were expressed in competent cells, such as E. coli BL21 (DE3), BL21-Gold (DE3), and Rosetta (DE3). Tag of target proteins were His-tag and GST-tag and the two truncated hTLT-2 proteins were purified with Ni-column and GSTrap FF column, respectively. Guanidine hydrochloride (Gdn-HCl) was used to dissolving the truncated hTLT-2 involved in inclusion body and the target proteins were refolded by dialysis for removing Gdn-HCl in the buffer. The results demonstrate that the larger size of 26-kDa of outer-membrane region of truncated hTLT-2 proteins are difficult to be expressed in E. coli expression system and tend to aggregate with other proteins in inclusion body. According to circular dichroism spectrum, the secondary structure of this 16-kDa of His-tagged hTLT-2 fragment is composed of about 5% alpha helix, 47% beta sheet, and 48% random coil. Thus, the secondary structure of outer-membrane region of 16-kDa of truncated hTLT-2 like TREM-1 and TLT-1 of TREM family mainly comprises beta sheets.