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Functional characterization and differential expression studies of squalene synthase from Withania somnifera

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Abstract

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.

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Abbreviations

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

SQS:

Squalene synthase

SQS :

Squalene synthase gene

TBME:

Tert-butyl methyl ether

qRT-PCR:

Quantitative real-time PCR

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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.

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Authors and Affiliations

  1. Plant Tissue Culture Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India

    Neha Gupta, Poonam Sharma, R. J. Santosh Kumar, Rishi K. Vishwakarma & B. M. Khan

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  1. Neha Gupta
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  2. Poonam Sharma
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  3. R. J. Santosh Kumar
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  4. Rishi K. Vishwakarma
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  5. B. M. Khan
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Correspondence to B. M. Khan.

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Neha Gupta and Poonam Sharma contributed equally to this study.

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Gupta, N., Sharma, P., Santosh Kumar, R.J. et al. Functional characterization and differential expression studies of squalene synthase from Withania somnifera . Mol Biol Rep 39, 8803–8812 (2012). https://doi.org/10.1007/s11033-012-1743-4

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