Kinetic model of in vivo folding and inclusion body formation in recombinantEscherichia coli

Abstract

Aggregation of misfolded proteins can reduce the yield in recombinant protein production. The underlying complex processes are additionally influenced by cellular physiology. Nevertheless, a lumped‐parameter model of kinetic competition between folding and aggregation was sufficient to track properly the specific concentration of a human protein produced in E. coli and its partitioning into soluble and insoluble cell fractions. Accurate estimation of the protein‐specific parameters required informative experiments, which were designed using the Fisher information matrix. The model was employed to calculate the influence of the specific glucose uptake rate in high‐cell‐density cultivation of E. coli on accumulation and aggregation of the recombinant protein. Despite its simplicity, the model was flexible and unbiased concerning unidentified mechanisms. Assuming an exponentially decreasing production rate, the irreversible aggregation step was found to follow firstorder kinetics, while assuming a constant production rate with simultaneous degradation, the model predicted transient aggregation only. Implications for strain and process development are discussed. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 315–322, 2001.

Bacterial Inclusion Bodies: Discovering Their Better Half

“…In vitro studies on protein aggregation indicated variability in the mechanisms of aggregation even for a given protein [8]. Kinetic analysis of in vitro protein aggregation [8][9][10] but also in vivo monitoring of IB formation [11] revealed that aggregate formation can be described as a pseudo-first-order process. There are two possible mechanisms for IB formation, either IBs start growing from single or limited number of molecules acting as nucleation site or from smaller aggregates which assemble and form larger aggregates [12] .…”

Investigating the Effects of Mutations on Protein Aggregation in the Cell

“…DISCUSSION Fig. 1 shows a simplified scheme describing the conformational opportunities for a protein such as HypF-N after biosynthesis in a bacterial cell (3,43). The protein is synthesized and released from the ribosome as an unfolded polypeptide chain.…”

“…The first early event of aggregation of HypF-N, which also occurs from the partially folded state, is also relatively rapid and takes place within 5 s (35,42). Changes of protein concentrations in an E. coli cell as a result of biosynthesis and degradation occur much more slowly, on the time scale of hours (43). This implies that although the total amount of HypF-N within a cell changes with time after induction of expression, the native, unfolded, partially folded, and initial aggregated states are in constant dynamic equilibrium.…”

Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli

“…Accumulation of soluble hFGF-2 starts with a delay after heatshock protein synthesis reached their peak level (Hoffmann and Rinas, 2000). Modelling data indicate that the soluble state might be reached only after in vivo refolding from the aggregated state (Hoffmann et al, 2001).…”