Purification and initial characterization of the lymphokine soluble immune response suppressor.
Two molecular forms of the lymphokine soluble immune response suppressor (SRIS), obtained from serum-free supernatant fluids of a T cell hybridoma producing SIRS, have been purified by a combination of gel filtration and high performance liquid chromatography (HPLC). Supernatant fluids (8 liters) from the hybridoma 393.D2.6 were concentrated and fractionated on Sephadex G-50 in 0.4 M pyridine/0.4 M acetic acid buffer. Active fractions containing approximately 1 X 10(7) units of SIRS activity and 300 mg protein were fractionated further by reverse phase HPLC on a Lichrosorb RP-18 column in a 1.0 M pyridine-0.5 M acetic acid buffer. A stepwise n-propanol gradient reproducibly eluted SIRS activity in the 20% n-propanol fraction. After three chromatography steps on the Lichrosorb RP-18 column, final purification was obtained with an Si-100 diphenyl column by employing the same buffer and elution system. SIRS eluted in the 30% n-propanol fraction as two discrete protein peaks. Purity of SIRS was assessed by SDS-polyacrylamide gel electrophoresis. The two protein peaks from the fractionation on the diphenyl column each exhibited only one band of protein with m.w. of 21,500 and 14,000, respectively. Additional studies of both molecular species of SIRS showed they possessed functional properties identical to those previously described for crude SIRS. Final purification of the 21,500 SIRS species yielded approximately 10 micrograms of protein and 6 X 10(8) units of SIRS activity or 6 X 10(10) units/mg protein; final purification of 14,000 SIRS species yielded approximately 30 micrograms of protein and a similar amount of SIRS activity or 2 X 10(10) units/mg protein.[1]References
- Purification and initial characterization of the lymphokine soluble immune response suppressor. Aune, T.M., Webb, D.R., Pierce, C.W. J. Immunol. (1983) [Pubmed]
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