CUMULATIVE SEDIMENTATION ANALYSIS OF ESCHERICHIA-COLI DEBRIS SIZE

A new method to measure Escherichia coil cell debris size after homoge nization is presented. It is based on cumulative sedimentation analysi s under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacr ylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins . The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method . Median debris size decreased significantly from approximately 0.5 mu m to 0.3 mu m as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cell s in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or in passes, possibly because of confounding intact cells and the exist ence of a minimum debris size for the conditions investigated. Recombi nant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure th e size distribution of inclusion bodies. This result compared extremel y well with an independent determination using centrifugal disc photos edimentation (CDS), thus validating the method. This is the first meth od that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. ex tinction coefficients), or the separation of debris and inclusion bodi es prior to analysis. (C) 1997 John Wiley & Sons, Inc.