TY - JOUR
T1 - Functional characterization and differential expression studies of squalene synthase from Withania somnifera
AU - Gupta, Neha
AU - Sharma, Poonam
AU - Santosh Kumar, R. J.
AU - Vishwakarma, Rishi K.
AU - Khan, B. M.
N1 - Funding Information:
Acknowledgments The authors thank Dr. H. V. Thulasiram, Organic chemistry, National Chemical Laboratory (Pune, India) for providing GC–MS facility; Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial support and University Grants Commission (UGC), New Delhi, India for providing fellowship.
PY - 2012/9
Y1 - 2012/9
N2 - Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.
AB - Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.
UR - https://www.scopus.com/pages/publications/84865150041
UR - https://www.scopus.com/inward/citedby.url?scp=84865150041&partnerID=8YFLogxK
U2 - 10.1007/s11033-012-1743-4
DO - 10.1007/s11033-012-1743-4
M3 - Article
C2 - 22718506
AN - SCOPUS:84865150041
SN - 0301-4851
VL - 39
SP - 8803
EP - 8812
JO - Molecular Biology Reports
JF - Molecular Biology Reports
IS - 9
ER -