[Rapid cloning and functional characterization of hypericin synthase gene].
Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, commonly known as St. John's wort. Hypericin has attracted a growing attention of the pharmaceutical industry because of its potential application to various therapies, including the treatment of depression and remarkable antiviral and photodynamic activities, hyp-1 gene encodes for phenolic coupling protein which catalyzes in vitro direct and specific conversion of emodin to hypericin which, however, has not formed common opinion so far. Six pairs of primers specific to hyp-1 gene were synthesized. The rapid cloning of hyp-1 gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pET32ahyp containing hyp-1 gene was constructed and was transformed into E. coli to induce heterologous expression. SDS-PAGE and Western blot results showed the recombinant Hyp-1 protein was expressed successfully in E. coli. The soluble fraction was used to test the function of the recombinant Hyp-1. Hypericin was detected by LC-MS/MS with emodin as a substrate under in vitro conditions. The above results corroborated the Hyp-1 function, a confusing question, which lay a material foundation for the synthesis of hypericin by synthetic biotechnology.
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
Yan-Wei
YW
Shi
Xiao-Hui
XH
Zhi
Hai-Na
HN
Zheng
Yan
Y
Yang
Wei
W
Wang
Jian-Mei
JM
An
Jian-Qiang
JQ
Kong
D000873:Anthracenes; D000928:Antidepressive Agents / Q000302:isolation & purification / Q000378:metabolism; D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism; D060326:Chemistry Techniques, Synthetic; D004642:Emodin / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005822:Genetic Vectors; D020902:Hypericum / Q000737:chemistry*; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010569:Perylene / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010946:Plants, Medicinal / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D000873:Anthracenes; D000928:Antidepressive Agents; D000998:Antiviral Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; D010569:Perylene; C004965:hypericin; D010453:Peptide Synthases; D004642:Emodin