Low expression temperatures have been successfully used in the past to increase the solubility of many proteins expressed in E. coli ; however, the molecular mechanisms GDC-0879 905281-76-7 responsible for this effect are not fully understood at present. The cold temperature protein chaperones are induced at low temperatures ; peptidyl-prolyl isomerase is a known cold temperature protein chaperone that catalyzes cis/trans isomerization of the peptide bonds found in proline residues. In addition, several ATP-consuming heat shock proteins may also play a role in improving protein solubility at low expression temperatures. Although highly inducible by heat shock treatment, these proteins are expressed at normal temperatures and have chaperone functions. However, the effects of lowering the expression temperature on protein solubility cannot be generalized because His6-tagged hGCSF was not soluble at all at 18uC. The effects of hGCSF purified from MBP-hGCSF or PDIb’a’- hGCSF on the proliferation of M-NFS-60 cells were slightly higher than that of commercially available hGCSF. The EC50 values for hGCSF purified from MBP-hGCSF and PDIb’a’-hGCSF were consistent with a previous study that reported an EC50 value in the range of 0.8–6 pM for hGCSF. At high concentrations, the purified hGCSF proteins induced mild inhibition of cell proliferation, resulting in a bellshaped biphasic dose-response curve. This is consistent with a previous report that other cytokines also show a biphasic dose-response curve. During DEM-assisted enucleation, DEM induces formation of a membrane protrusion that contains a mass of condensed maternal chromosomes, which can be easily removed with minimal damage. This simple method may prove useful for nuclear transfer and has garnered much attention. Here, we assessed changes in the level of MPF and the distribution of cyclin B1 in porcine oocytes following treatment with DEM. We also compared the efficiencies of DEMassisted and mechanical enucleation, and the development of embryos generated from these enucleated oocytes by SCNT. Our previous study demonstrated that brief treatment of MII porcine oocytes with DEM produces a membrane protrusion that contains a mass of condensed chromosomes, as seen in DEMtreated bovine oocytes and nocodazole-treated rat oocytes. Although the mechanisms by which DEM elicits its effects are unclear, condensation of maternal chromosomes might cause the protrusion to develop. Yin et al. reported that following treatment with 0.4 mg/ml DEM and 0.05 M sucrose for 1, 3, 6, 12, or 24 h, chromosomes were condensed in 100%, 97%, 87%, 96%, and 74% of porcine oocytes, respectively. In this previous study, all oocytes with condensed chromosomes had a protruding membrane, whereas oocytes with dispersed chromosomes did not. Such protrusions are frequently observed in DEMtreated bovine and rabbit oocytes. Sucrose does not affect the formation of a cytoplasmic protrusion containing chromosomes in pig oocytes, but does enlarge the perivitelline space. Therefore, the current study investigated the effects of DEM treatment on oocytes.