Purification And Characterization Of A Thymus Activity Factor I(TAF-I)from Calf Thymus

A new thymus activity factor I (TAF-I)was isolated and purified from calf thymus.The physico-chemical characters and biological activity of TAP- I were also studied in the work.The crude extract was obtained through homogenize,centrifugation and ultrasonicati-on from calf thymus.Then the crude extract was applied to Sephadex G-15 gel column (1.5 cmX 100cm),eluted with deionized water at flow rate 48mL/h,received 6 protein peak,peak II with E-rosette-activity was pooled and concentrated.Next,the peak II was chromatograp-hed on a DEAE-Sephadex A-25 column(1.5cmX 10cm),0.05~0.5moL/L ph5.8 NH4Ac-H-Ac buffer linear gradient elusion was carried out.Peak 6 with high E-rosette-activity was collected and lyophilized.This fraction was dissolved in deionized water and gel-filtrated on a Sephadex G-10 column(0.8cm X 45cm).The first peak was collected and lyophyliz-ed.Finally, the peak was chromatographed on revesed-phase HPLC(ZORBAX Extend-C18 column,4.6mm X 250mm), eluted with a linear gradient of 0%~50% acetonitrile in 0.1% trifluoroacetic acid in water,at the flow rate 0.06moL/L, The eluted peak at 10.46min was collected and lyophilized.0.92g TAF-I was obtained from 500g calf thymus tissue,the recovery yield was 102%.TAF-I exhibited the mass of molecule 618.2 analyzed by ESI-MS.Ultraviolet absorption spectra and FT-IR spectroscopy showed TAF-I was a peptide.Amino acid composition analysis showed that this peptide consisted of alanine,glycine,glutamic acid,glutamine, lysine and serine residues. TAF-I was sequenced by ESI tandem MS" to obtain the amino acid sequence.The accuracy and resolution of ESI-MS for determination and structural analysis of peptides and proteins are both better than that of the routine method.The results of E-rosette assay and MTT cdorimetric assay showed that TAF-I specifically increased E-rosette formation of human peripheral blood lymphocytes and increased the proliferative respones of the mitogen ConA-induced mice splenocytes,the effect of TAF-I was dose dependent.Compared with thymopeptidum, TAF-I has an samiliar enhancing effect in low dose.