Heterologous Expression and Purification of Auxin Influx Transporter AUX1 from Yeast

• Main Authors: Lee, Tzi Min, 李姿敏
• Other Authors: Pan, Rong Long
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/38301927066081852532

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 103 === Auxin is not only the first-identified plant growth hormone but also the most important hormone in plants. There are three types of natural auxins, including IAA, 4-Cl-IAA and IBA. Especially, IAA is the major type plant hormone. It regulates the growth and development of plants. The tactic used to maintain the homeostasis of auxin is crucial. It includes many aspects, such as synthesis, inactivation, degradation and transport of auxin. Moreover, auxin conforms to the traditional definition of hormone that substance needs to transport from source to sink. Therefore, AUX1, one of the auxin influx transporters, plays a significant role in maintaining the homeostasis of auxin. The molecular mass of AUX1 is about 54 kDa and the number of predicted transmembrane domains is 11. The study here focuses on the overexpression of Arabidopsis AUX1 in the budding yeast Saccharomyces cerevisiae (BJ2168, BY4741), and fission yeast Schizosaccharomyces pombe (leu1-32 h-, avt3). The specific expression systems contain different yeast strains and shuttle vectors, pYES2 (BJ2168, BY4741), pDR196 (BY4741) and pREP41 (avt3, leu1-32 h-). Conditions influencing the expression of protein were explored for the mass production of AUX1. The fractions with highest yield were obtained, followed by the detergent DDM (n-Dodecyl-β-D-maltopyranoside) treatment to isolate the protein from membrane. The metal chelating affinity chromatography was then used to purify the specific protein, AUX1. The 3D structure of AUX1 is expected to be investigated eventually.