Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins

A method to quantify the impact of proteolysis on accumulation of recombina nt proteins in E. coli is described. A much smaller intracellular concentra tion of staphylococcal protein A (SpA) (14.7 mg.g(-1)) compared to the fusi on protein SpA-beta galactosidase (138 mg.g(-1)) is explained by a very hig h proteolysis rate constant of SpA. The SpA synthesis rate reached a maximu m one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the Ist order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further S pA accumulation in cells, though synthesis continued for at least 10 h. Sim ilar experiments with recombinant protein ZZT2 also revealed that most of t he synthesized product was degraded. The order of proteolysis kinetics depe nded on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while a t high concentrations ZZT2 was degraded according to zero order kinetics. I n a protease Clp mutant the degradation rate decreased and intracellular co ncentration of ZZT2 increased from 50 mg.g(-1) to 120 mg.g(-1). The measure ments of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization . In this case the concentration would have increased from 50 to 280 mg.g(- 1) in II h. Thus, this method reveals the potential to increase the product ivity by eliminating proteolysis. (C) 2000 Elsevier Science Inc. All rights reserved.