Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays.
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 pTyr(525)/Tyr(526) 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.
Singh
Deepika
D
Department of Biotechnology and Bioinformatics, Ranbaxy Research Laboratories, Gurgaon, Haryana, India. deepika.singh@ranbaxy.com
Rani
Reema
R
Rajendran
Resmi
R
Kaur
Namrata Jit
NJ
Pandey
Abhinav
A
Chopra
Puneet
P
Jain
Tarun
T
Jain
Manish Kumar
MK
Grover
Sonam
S
Arya
Ranjana
R
Saini
Kulvinder Singh
KS
D016367:Baculoviridae / Q000235:genetics*; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics* / Q000821:virology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000821:virology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000072377:Syk Kinase
D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase