Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays

Deepika Singh, Reema Rani, Resmi Rajendran, Namrata Jit Kaur, Abhinav Pandey, Puneet Chopra, Tarun Jain, Manish Jain, Sonam Grover, Ranjana Arya, Kulvinder Singh Saini

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.

Original languageEnglish (US)
Pages (from-to)201-212
Number of pages12
JournalBiotechnology Journal
Volume5
Issue number2
DOIs
StatePublished - Feb 2010
Externally publishedYes

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Keywords

  • Expression
  • Phosphorylation
  • Purification
  • Recombinant
  • Syk

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

Cite this

Singh, D., Rani, R., Rajendran, R., Kaur, N. J., Pandey, A., Chopra, P., Jain, T., Jain, M., Grover, S., Arya, R., & Saini, K. S. (2010). Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays. Biotechnology Journal, 5(2), 201-212. https://doi.org/10.1002/biot.200900139