Cellular response to accumulation of recombinant proteins in the E. coli inner membrane: Implications for proteolysis and productivity of the secretory expression system (original) (raw)
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Recombinant protein secretion in Escherichia coli
Biotechnology Advances, 2005
The secretory production of recombinant proteins by the Gram-negative bacterium Escherichia coli has several advantages over intracellular production as inclusion bodies. In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding, and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost. This review presents several strategies that can be used for recombinant protein secretion in E. coli and discusses their advantages and limitations depending on the characteristics of the target protein to be produced.
Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins
Enzyme and Microbial Technology, 2000
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 ⅐ g Ϫ1 ) compared to the fusion protein SpA-galactosidase (138 mg ⅐ g Ϫ1 ) 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 ⅐ g Ϫ1 to 120 mg ⅐ g Ϫ1 . 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 ⅐ g Ϫ1 in 11 h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.
Applied Microbiology and Biotechnology, 2005
The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P malK promoter; and the cytoplasmic part of the production was compared with production from the P lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.
Microbiological Research, 2001
Cell growth and production of recombinant proteins in stationary phase cultures of Escherichia coli recover concomitantly with spontaneous lysis of a fraction of the ageing cell population. Further exploration of this event has indicated that sonic cell disruption stimulates both cell growth and synthesis of plasmid-encoded recombinant proteins, even in exponentially growing cultures. These observations indicate an efficient cell utilisation of released intracellular material and also that this capability is not restricted to extreme nutrient-starving conditions. In addition, the efficient re-conversion of waste cell material can be viewed as a potential strategy for an extreme exploitation of carbon sources and cell metabolites in production processes of both recombinant and non-recombinant microbial products.
Applied Microbiology and Biotechnology, 2003
This paper is a review of strategies to introduce protein into the liquid medium of Escherichia coli K-12 industrial production cells. The cell design strategies are generally based on one of two general mechanisms. The first strategy involves a two-stage translocation using active transporters in the cytoplasmic membrane followed by passive transport through the outer membrane. Passive transport is achieved through either external or internal destabilization of the E. coli structural components. The latter can be achieved by transplantation of destabilizing components (lysis proteins) that work by permeabilization of the outer membrane from the interior of the cell, or by using cells carrying mutations of structural components. Passive transport can also be achieved by a chemical, mechanical, or enzymatic permeabilization directed from outside the cell. The second strategy is realized through transplantation of proteins capable of active transport over one or both of the membranes. This involves the transplantation of secretion mechanisms into the K-12 cell from pathogenic E. coli as well as from other species. The process design strategies are dependent on environmental conditions and must take into account changes in physical parameters, medium design, and influx of limiting carbon source in fed-batch cultivation.
Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli
Microbial cell factories, 2005
Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubil...
Analysis of factors affecting the periplasmic production of recombinant proteins in Escherichia coli
Journal of microbiology and biotechnology, 2007
Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form alpha-helix structures are more translocated to the periplasm.