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 amounts of individual contaminants were added to test their effect on renaturation 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 reaction, but some of them significantly affected the rate of the aggregation 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 decrease of about 10% in the overall renaturation yield. Phospholipids were found to improve refolding yields by about 15% by decreasing the overall 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 found to significantly decrease renaturation yields by promoting aggregation. This effect was strongly dependent on the concentration of the proteinaceous impurity. On the other hand, the presence of refolding ribonuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation kinetics of lysozyme, even at concentrations as high as 0.7 mg/mL.
Maachupalli-Reddy
J
J
Department of Chemical Engineering, Tufts University, Medford, Massachusetts 02155, USA.
Kelley
B D
BD
De Bernardez Clark
E
E
D001426:Bacterial Proteins / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004527:Egg Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000737:chemistry*; D010957:Plasmids; D011489:Protein Denaturation; D017510:Protein Folding; D012335:RNA, Ribosomal / Q000737:chemistry*
D001426:Bacterial Proteins; D004527:Egg Proteins; D008070:Lipopolysaccharides; D010743:Phospholipids; D012335:RNA, Ribosomal; D004247:DNA; D009113:Muramidase