Expression, purification, phosphorylation and characterization of recombinant human statherin.
This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria-Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47mg per liter of cell culture, while 112mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP-HPLC corresponded to 0.6mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.
Manconi
Barbara
B
Dipartimento di Scienze Applicate ai Biosistemi, Sezione di Biochimica e Biologia Molecolare, Università di Cagliari, Cagliari, Italy.
Cabras
Tiziana
T
Vitali
Alberto
A
Fanali
Chiara
C
Fiorita
Antonella
A
Inzitari
Rosanna
R
Castagnola
Massimo
M
Messana
Irene
I
Sanna
Maria Teresa
MT
D000818:Animals; D047388:Casein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D010306:Parotid Gland / Q000166:cytology; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013552:Swine
D011993:Recombinant Fusion Proteins; C018149:STATH protein, human; D012471:Salivary Proteins and Peptides; D047388:Casein Kinases