臺灣博碩士論文加值系統

Abstract
The metal-responsive transcription factor-1 (MTF-1) protein is a highly conserved zinc finger protein that is essential for activation of metallothionein genes in response to heavy metals. Although phosphorylation of MTF-1 has been found to regulate expression of MT genes, the other post-translational modifications of this protein has not been described. Herein, we found that MTF-1 could undergo sumoylation by SUMO-1 in E. coli. Using co-transformation of pGEX-MTF-1 wild type (WT) or its sumoylation deficient versions (MTF-1 K14R, K27R and K14R/K627R) with pT-E1/E2/S1-His, a plasmid expressing Aos1, Uba2, Ubc9 and SUMO-1, we identified lysine 627 within 626IKQE629 motif was the major sumoylation acceptor site whereas lysine 14 within 13FKGE16 motif, was the minor one. We also revealed that the unmodified MTF-1 could not be separated completely from the sumoylated form by using a range of purification methods. Therefore, other methods should be employed for purification of the sumoylated protein. Finally, we have initially established the condition of electromobility shift assay (EMSA) for in vitro DNA binding activity of MTF-1 and sumoylated MTF-1, if any, to MRE promoter.
Key words: MTF-1, Sumoylation, Purification of sumoylated protein, MRE, EMSA.

Table of contents
Abstract 1
中文摘要 2
Chapter 1 Introduction 3
1.1. Metal-responsive transcription factor 1 (MTF-1) 3
1.2. Sumoylation 5
Chapter 2 Materials and methods 10
2.1. Chemicals 10
2.2. Construction of protein expression plasmids 10
2.3. Preparation of competent cell 11
2.4. Recombinant protein expression and purification 11
2.5. Combination of different purification techniques 13
2.6. Digestion of GST tag by PreScission Protease 13
2.7. Purification of protein with TALON metal affinity resin 14
2.8. Western blotting 14
2.9. Coomassie brilliant blue staining 15
2.10. Electromobility shift assays (EMSAs) 15
Chapter 3 Results 17
3.1. Expression of sumoylated MTF-1 in Escherichia coli 17
3.2. Mapping the MTF-1 sumoylation site(s) 20
3.3. Purification of sumoylated MTF-1 in Escherichia coli 21
3.4. DNA binding activity assay 24
Chapter 4 Discussion 26
References 32

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