Comparison of Natural and Artificial Split Intein in Resisting Detergents and Denaturants

• Main Author: 盧佑成
• Other Authors: 蘇士哲
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/07858178177635321348

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 102 === The protein splicing is an autocatalytic protein ligation process by intein. The protein splicing ligates the N- and C-terminal flanking sequence, termed “extein”, and excised intein domain itself. The process comes in two flavors. Splicing reaction in single polypeptide chain from one component in terms of cis-splicing. By contrast, the trans-splicing associated two components by using split inteins. Furthermore, the PTS not only occur in vivo but also enable in vitro. Until recently, the biological application of protein trans-splicing (PTS) includes site-specific incorporation of biophysical probes, segmental isotope labeling for NMR structure study or dynamics research, protein semi-synthesis containing posttranslational modifications, and protein cyclization. PTS also elicits a possibility from segmental isotope labeling and macromolecular protein structure determination by solution NMR. To precede the PTS reactions under different environments becomes a concerned issue for split intein. The previous data indicated that nature Nostoc punctiforme (Npu) DnaE split intein (Npu102) has great tolerance in the presence of either detergents or denaturants. In our study, we examined trans-splicing activity of a newly engineering Npu DnaE split intein (Npu36) in the presence of urea, guanidine hydrochloride or detergents. In comparing to Npu102, the N-terminal intein fragment of Npu36 was shorter than that in Npu102. Therefore, the short N-terminal portion provides benefit in PTS if synthesis of N-terminal intein fragment is required. In the study, we demonstrate the hydrogen bonds and charge-charge interaction between N- and C-terminal intein fragments of split intein make significant contributions than overall structure of split intein for PTS.