Enhanced yield and homogeneity of buffalo growth hormone by an improved chromatographic protocol

Abstract

Loss of buffalo Growth Hormone (buGH) in the various side fractions of standard buGH purification protocol has been determined quantitatively by direct binding ELISA and qualitatively by SDS-PAGE and Western blot analysis. Accounting result indicated that there was a considerable loss of buGH in the side fractions. An alternative protocol to prevent loss and to obtain a high yield of buGH has been developed by introducing anion exchange chromatography, QAE-Sephadex. This has resulted in a simple, reproducible three-step protocol. In this protocol, an extract obtained at 250 mM (NH₄)₂ SO₄, pH 5.5, was loaded onto the QAE-Sephadex column in 0.1 M NH₄ HCO₃. At this salt concentration, the bulk of the buGH came as QAE unbound fraction. Some amount of buGH, together with contaminating proteins, was bound to QAE-Sephadex and these could be eluted with 1 M KCl. The immunopotency of the enriched buGH preparation "QUB" (QAE unbound fraction) in a direct binding ELISA was similar to that of the semi-pure buGH (ECS/APECS) preparation obtained using the standard protocol, but the yield was 4 times higher. The SDS-PAGE data showed that the banding pattern of standard semi-pure buGH and QUB were quite similar and QUB can be loaded onto the Sephacryl S-200 gel filtration chromatography to yield a highly purified buGH. SDS-PAGE and Western blot analyses showed the major band of buGH in QUB at the same position as in the case of standard buGH. It has also been demonstrated here that it is possible to separate buffalo prolactin (buPRL) and buGH on QAE-Sephadex.