Mxr1磷酸化水平受Ptp调控机理的初步研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-0020

生物技术通报 ›› 2019, Vol. 35 ›› Issue (7): 108-113.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0020

Mxr1磷酸化水平受Ptp调控机理的初步研究

侯成林, 杨艳坤, 陈嘉荔, 白仲虎

江南大学生物技术学院碳水化合物与生物技术教育部重点实验室工业生物技术教育部重点实验室谷物发酵技术国家工程实验室,无锡 214122

收稿日期:2019-01-08 出版日期:2019-07-26 发布日期:2019-07-29
作者简介:侯成林,硕士研究生,研究方向：酵母细胞信号通路;E-mail：m18851505776@163.com
基金资助:
国家自然科学基金项目（21808082,21878124）,江苏省研究生科研创新计划项目（KYCX18_1803,KYCX18_1904）

Preliminary Study on the Regulation of Mxr1 Phosphorylation by Ptp

HOU Cheng-lin, YANG Yan-kun, CHEN Jia-li, BAI Zhong-hu

The Ministry of Education Key Laboratory of Carbohydrate Chemistry and Biotechnology,School of Biotechnology,Jiangnan University; The Ministry of Education Key Laboratory of Industrial Biotechnology,School of Biotechnology,Jiangnan University; National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122

Received:2019-01-08 Published:2019-07-26 Online:2019-07-29

摘要/Abstract

摘要： 通过PULL-DOWN找到与巴斯德毕赤酵母转录激活因子Mxr1相互作用的小分子酪氨酸磷酸酶Ptp,并验证其去磷酸化功能,可以使磷酸化的Mxr1第215位丝氨酸的磷酸基团水解。用Mxr1第215位特定位点磷酸化抗体Western blot检测Mxr1S215在不同培养基中磷酸化情况。发现野生菌株中Mxr1在甘油培养基没有磷酸化,在甲醇培养基发生磷酸化。在Ptp高表达菌株中,无论在甘油还是甲醇培养基Mxr1都没有发生磷酸化,而在Ptp敲除菌株中磷酸化明显高于野生型菌株,说明Ptp调控Mxr1磷酸化。在大肠杆菌中表达并纯化,测定了Ptp酶学性质。构建了巴斯德毕赤酵母Mxr1S215A突变株,发现多个与甲醇代谢相关的基因在转录水平发生变化,推测其可能受S215位点磷酸化Mxr1的调控。

关键词: 巴斯德毕赤酵母, 转录激活因子Mxr1, 磷酸酶, 磷酸化

Abstract: The small molecule tyrosine phosphatase Ptp interacting with Pichia pastoris transcriptional activator Mxr1 was found by PULL-DOWN,and its function of dephosphorylation was verified,resulting in the hydrolysis of phosphate group in phosphorylated Mxr1 215th serine. Phosphorylation of Mxr1S215 in different media was detected by Western blot of Mxr1S215th specific site phosphorylation antibody. It was found that Mxr1in the wild strain was not phosphorylated in the glycerol medium,while phosphorylated in the methanol medium. In the Ptp-highly expressed strain,no phosphorylation occurred in Mxr1 in either glycerol or methanol medium,but phosphorylation was significantly higher in the Ptp-knockout strain than in the wild type one,indicating that Ptp indeed regulated Mxr1 phosphorylation. The expression in Escherichia coli was conducted,then the proteins were purified,and the Ptp enzymatic properties were determined. The P. pastorisMxr1S215A mutant strain was constructed,and several genes related to methanol metabolism were found to change at the transcriptional level,suggesting that it was regulated by Mxr1 phosphorylation at 215th site.

Key words: Pichia pastoris, transcriptionalactivator Mxr1, phosphatase, phosphorylation

侯成林, 杨艳坤, 陈嘉荔, 白仲虎. Mxr1磷酸化水平受Ptp调控机理的初步研究[J]. 生物技术通报, 2019, 35(7): 108-113.

HOU Cheng-lin, YANG Yan-kun, CHEN Jia-li, BAI Zhong-hu. Preliminary Study on the Regulation of Mxr1 Phosphorylation by Ptp[J]. Biotechnology Bulletin, 2019, 35(7): 108-113.

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Mxr1磷酸化水平受Ptp调控机理的初步研究

Preliminary Study on the Regulation of Mxr1 Phosphorylation by Ptp

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