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

Abstract

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg. g(-1)) compared to the fusion protein SpA-betagalactosidase (138 mg. g(-1)) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum 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 1st order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg. g(-1) to 120 mg. g(-1). The measurements 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 11 h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.