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Degradation kinetics of an abnormal beta-galactosidase in Escherichia coli

Citation

Kosinski, Michael J. (1991) Degradation kinetics of an abnormal beta-galactosidase in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06222007-141447

Abstract

Many recombinant proteins expressed in Escherichia coli are identified by the cell as abnormal and are degraded rapidly. As a model protein, we employed an unstable fragment of E. coil beta-galactosidase, the CSH11 mutant, coded by a single copy in the chromosome. This experimental system enabled identification of the process and cellular variables having the greatest impact on the degradation rate of abnormal proteins.

The in vivo degradation rates were measured using pulse-chase radioactive labeling techniques, and intracellular concentrations were determined using [alpha]-complementation assays. The degradation of the CSH11 fragment was sensitive to the culture temperature and approximately followed Arrhenius behavior, but the apparent degradative rate constant for its 90 kDa degradative intermediate increased by an order of magnitude between 37 and 40°C. The apparent rate constants for both beta-galactosidase fragments inexplicably decreased with increasing induction level. There was no aggregation of the fragments to account for this decrease in rate constants.

Cellular protease levels were determined using an in vitro assay of cell extracts. The ATP-independent proteases were not induced when the abnormal beta-galactosidase was expressed, and their level decreased by 20% from 37° to 42°C. Expression of the abnormal protein caused a doubling in the ATP-dependent proteolytic activity. This ATP-dependent activity also increased 2.5-fold from 30° to 42°C with induction of the abnormal protein.

Stress (heat shock-like) responses were detected by radioactive pulse- labeling of cellular proteins and analysis by one- and two-dimensional PAGE. There was no response to induction of the abnormal beta-galactosidase at 30°C, even though the concentration of abnormal protein was approximately the same as at 37°C where a stress response was observed. Determined with the protease assay, ATP-dependent proteolytic activity was associated with the stress proteins independent of protease La.

Proteolytic susceptibility to [alpha]-chymotrypsin correlated well with the thermal stability of the affinity purified, beta-galactosidase fragments. Thermal instability of the 90 kDa intermediate is the likely cause of the significant increase in its in vivo degradation rate between 37° and 40°C.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemical Engineering
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Bailey, James E.
Thesis Committee:
  • Unknown, Unknown
Defense Date:22 May 1991
Record Number:CaltechETD:etd-06222007-141447
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06222007-141447
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2692
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:19 Jul 2007
Last Modified:26 Dec 2012 02:53

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