Development of the method for large-scale preparation of bioactive cannabinoids

大规模制备生物活性大麻素方法的开发

• 批准号: 10672108
• 负责人: MORIMOTO Satoshi
• 金额: $ 1.73万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672108/
• 关键词: Marihuana; cannabinoid; THCA synthase; CBDA synthase; CBCA synthase; cDNA cloning; PCR; large-scale preparation

In this study, I attempted to develop method for large-scale preparation of three bioactive cannabinoids, THCA, CBDA and CBCA using biotechnology.As a first step, purified THCA synthase and CBDA synthase were digested with CNBr or protease, ant the resulting peptide fragments were N-terminally sequenced. The various primers were designed based on these amino acid sequences, together with N-terminal amino acid sequences of both enzymes. Concerning the cDNA templates, mRNA was prepared from the Mexican Cannabis strain (for cloning of THCA synthase) and CBDA Cannabis strain (for cloning of CBDA synthase), and cDNA was synthesized from each mRNA preparation by reverse transcriptase reaction. Cloning of genes encoding THCA synthase and CBDA synthase was achieved by degenerate PCR, followed by 3'- and 5'-RACE. Their genes were found to consist of 1635- and 1632- nucleotide open reading frames encoding proteins of 545 and 544 amino acid residues, respectively. The deduced amino acid sequences of THCA synthase and CBDA synthase showed high identity (84%) to each other and moderately high identity (40%) to berberine bridge enzyme.I attempted development of expression systems for THCA synthase and CBDA synthase using the cloned genes. The regions coding deduced mature THCA synthase and CBDA synthase were amplified by PCR, and the resulting cDNAs were incorporated into the expression vector pET28a. After transformation of E. coli JM109, expression of the recombinant protein was induced by the addition of isopropyl-l-β-D-thiogalactoside. The expression of recombinant proteins was confirmed, whereas no proteins had cannabinoid synthase activity. Hence, pYE22m was used as a expression vector. Consequently, I confirmed the expression of catalytically active THCA synthase in yeast. Now, for the expression of CBDA synthase, similar experimet using pYE22m is just started.

本研究尝试利用生物技术大规模制备三种大麻素THCA、CBDA和CBCA，首先将纯化的THCA合成酶和CBDA合成酶用CNBr或蛋白酶消化，并对得到的肽段进行N端测序。基于这些氨基酸序列以及两种酶的N-末端氨基酸序列设计各种引物。关于cDNA模板，从墨西哥大麻株（用于克隆THCA合酶）和CBDA大麻株（用于克隆CBDA合酶）制备mRNA，并通过逆转录酶反应从每个mRNA制备物合成cDNA。通过简并PCR，随后通过3 '-和5'-RACE实现编码THCA合酶和CBDA合酶的基因的克隆。它们的基因分别由1635和1632个核苷酸的开放阅读框组成，分别编码545和544个氨基酸残基的蛋白质。推导的THCA合成酶和CBDA合成酶的氨基酸序列彼此之间具有较高的同源性（84%），与小檗碱桥酶的同源性也较高（40%）。通过PCR扩增推导的成熟THCA合酶和CBDA合酶的编码区，并将所得cDNA整合到表达载体pET 28 a中。转化E. coli JM 109中，加入异丙基-1-β-D-硫代半乳糖苷诱导表达。重组蛋白的表达得到证实，但没有蛋白质具有大麻素合成酶活性。因此，使用pYE 22 m作为表达载体。因此，我证实了催化活性THCA合酶在酵母中的表达。现在，对于CBDA合酶的表达，使用pYE 22 m的类似实验才刚刚开始。