Byungyun Cho

Purification of inclusion body—forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins

Proteins and peptides expressed in the prokaryotic system often form inclusion bodies. Solubilization and refolding procedures can be used for their recovery, but this process remains difficult. One strategy for improving the solubility of a protein of interest is to fuse it to a highly soluble protein. To select a suitable fusion partner capable of solubilizing the aggregation-prone (inclusion body-forming) proteins and peptides, Escherichia coli thermostable proteins were identified and tested. Among them, trigger factor (TF) protein was selected because of its high expression and stability. Using an expression system based on fusion to TF, selected proteins and peptides that otherwise form inclusion bodies were expressed in soluble state and were purified like other soluble proteins. This system provides a convenient method for production of aggregation-prone proteins and peptides.

Efficient cleavage of Bid and procaspase-7 by caspase-2 at lower pH.

The activity of caspase-2 was examined under varying biochemical conditions with the synthetic and protein substrates, Bid and procaspase-7. The results indicate that it was largely influenced by pH which might be one reason behind the inconsistency for the cleavage of its established substrates during caspase-2-induced apoptosis.

Purification of catalytically active caspase-12 and its biochemical characterization.

Caspase-12, mainly detected in endoplasmic reticulum (ER), has been suggested to play a role in ER-mediated apoptosis and inflammatory caspase activation pathway. Cleavage of the prodomain by caspase-3/-7 at the carboxyl terminus of Asp94 or m-calpain at the carboxyl terminus of Lys158 was reported to be a part of caspase-12-involved apoptosis. We biochemically characterized the prodomain-free forms of caspase-12 and the equivalent enzymes; Deltapro1(G95-D419), rev-Deltapro1[(T319-N419)-(G95-D318), a reverse form of Deltapro1] and rev-Deltapro2[(T319-N419)-(T159-D318)]. The three variants showed comparable activities which were dependent on salt concentration and pH. Auto-proteolytic cleavage was observed at two sites (carboxyl termini of Asp318 and Asp320) in Deltapro1. Constitutively active forms of caspase-12 (rev-Deltapro1 and rev-Deltapro2) could induce cell death in cells transfected with the corresponding expression vectors, but no cleavage of caspase-3, DFF45 or Bid was observed, indicating caspase-12 may mediate a distinct apoptotic pathway rather than caspase-8 or -9-mediated cell death.

Comparative Study of Apoptosis Induced by H2O2 and NO: Limitation of Apoptosis Induced by NO Due to Slower Recovery of Activity of NOModified Caspase

Both H(2)O(2) and NO can act as apoptogens, triggering apoptosis in many cells. They are also well known inhibitors of caspases, essential enzymes in apoptosis. The differences between these two agents as apoptosis inducers and how caspases mediate apoptosis with these inhibitory agents is still unclear. Consistent with the previous reports, these two agents induced apoptosis accompanied by caspase activation with limitation of all apoptotic events for NO. It was found that NO-modified caspase-3 showed a slower recovery of its activity in the presence of the reducing agents compared to that of H(2)O(2) modification. This is one possible cause of the limited apoptosis in the case of NO.

Effects of high pressure on the magnetic properties of Eu1-xCaxB6Eu1-xCaxB6 (x=0.3x=0.3, 0.35, and 0.4)

We present our study of Ca substituted hexaborides, Eu1-xCaxB6Eu1-xCaxB6 (x=0.3x=0.3, 0.35, and 0.4), through temperature-dependent magnetization M(TM(T) and isothermal magnetization M(H)M(H) measurements under high pressures (0⩽p⩽10kbar) and magnetic fields (0⩽H⩽7T). TCTC of x = 0.3 increases with increasing pressure and the ferromagnetic interaction is observed to be increased in M(H)M(H). The antiferromagnetic transition behavior seen in x=0.4x=0.4 at ambient pressure is gradually weakened with increasing pressure and completely subdued at p=10kbar. From these results, we found that the physical properties of Eu1-xCaxB6Eu1-xCaxB6 are dominated by the effective magnetic interaction between Eu moments.