Molecular cloning and functional analysis of squalene synthase (SS) in Panax notoginseng.

Panax notoginseng (Burk.) F. H. Chen, which is a used traditional Chinese medicine known as Sanqi or Tianqi in China, is widely studied for its ability to accumulate the triterpene saponins. Squalene synthase (SS: EC 2.5.1.21) catalyzes the first enzymatic step from the central isoprenoid pathway toward sterol and triterpenoid biosynthesis. In this study, SS from P. notoginseng was cloned and investigated followed by its recombinant expression and preliminary enzyme activity. The nucleotide sequence of the ORF contains 1 248 nucleotides and encodes 415 amino acid residues with molecular weight of 47.16kDa and pI of 6.50. Bioinformatics analysis revealed that the deduced PnSS protein had a high similarity with other plant squalene synthases. To obtain soluble recombinant enzymes, 29 hydrophobic amino acids were deleted from the carboxy terminus and expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3). Approximately 66.46kDa recombinant protein was checked on SDS-PAGE and Western Blot analysis. Preliminary activity of the resultant bacterial crude extract was analyzed by gas chromatograph-mass spectrometer (GC-MS). The identification and function of PnSS is important for further studies of the triterpene saponins biosynthesis in P. notoginseng.