EFFECT OF INCLUSION-BODY CONTAMINANTS ON THE OXIDATIVE RENATURATION OF HEN EGG-WHITE LYSOZYME

The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate and yield of hen egg white lysozyme was investigated. Separate experiments were conducted in which known amoun ts of individual contaminants were added to test their effect on renat uration :kinetics. On the basis of a simplified model for the kinetic competition between folding and aggregation, it was found that none of the above contaminants had an effect on the rate of the folding react ion, but some of them significantly affected the rate of the aggregati on reaction and, thus, the overall renaturation yield. While ribosomal RNA did not seem to affect the aggregation reaction, plasmid DNA and lipopolysaccharides increased the aggregation rate, resulting in a dec rease of about 10% in the overall renaturation yield.; Phospholipids w ere found to improve refolding yields by about 15% by decreasing the o verall rate of the aggregation reaction without affecting the rate of the folding reaction. Proteinaceous contaminants which aggregate upon folding, such as beta-galactosidase and bovine serum albumin, were fou nd to significantly decrease renaturation yields by promoting aggregat ion. This effect was strongly dependent on the concentration of the pr oteinaceous impurity. On the other hand, the presence of refolding rib onuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation k inetics of lysozyme, even at concentrations as high as 0.7 mg/mL.