Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli

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.     

Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli

We previously characterized a defective-folding mutant of maltose-binding protein of Escherichia coli, MalE31, which formed periplasmic inclusion bodies. Here, we show that MalE31 aggregation does not affect bacterial growth at 30 degrees C but is lethal at 37 degrees C. Surprisingly, under mild heat shock conditions at 42 degrees C, inclusion bodies are degraded and bacterial growth is restored. One physiological consequence for the cells overproducing MalE31 was to induce an extracytoplasmic stress response by increasing the expression of the heat shock protease DegP via the CpxA/CpxR two-component signalling pathway. Furthermore, we show that the Cpx response is required to rescue the cells from the toxicity mediated by MalE31. Finally, expression of highly destabilized MalE variants that do not aggregate in the periplasm also induces the Cpx pathway, indicating that inclusion body formation is not necessary to activate this specific extracytoplasmic stress regulatory system.     

A comparison of high cell density fed-batch fermentations involving both induced and non-induced recombinant Escherichia coli under well-mixed small-scale and simulated poorly mixed large-scale conditions

In this work, multi-parameter flow cytometric techniques, coupled with dual colour fluorescent staining, have been used to study the metabolic consequences of inclusion body formation in high cell density fed-batch cultures of the recombinant E. coli strain MSD3735, producing the IPTG inducible model mammalian protein, AP50. Further, we report on the development of the scale-down, two compartment (STR + PFR) experimental simulation model to study, for the first time, the effect of a changing microenvironment with respect to three of the major spatial heterogeneities that may be associated with large-scale bioprocessing (pH, glucose and dissolved oxygen concentration) on a recombinant bacterial system. Using various time points for induction and various scale-down configurations, it has been shown that inclusion body formation is followed immediately by a detrimental progressive change in individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability, resulting in a lower final biomass yield. However, the extent of this change was found to be dependent on whether the AP50 protein was induced or not, on the time of induction and on which combination of heterogeneities was being simulated. From this and previous work, it is clear that the scale-down two-compartment model can be used to study the impact of genetically modifying an organism to produce inclusion bodies and any range and combination of potential heterogeneities known to exist at the large scale.     

Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase

Recombinant mammalian proteins expressed in E. coli can be difficult to purify in high yield in a soluble and functional form. Various techniques have been described to prevent proteolysis of expressed proteins and/or their sequestering as insoluble aggregates within inclusion bodies. We report conditions for expressing recombinant proteins from E. coli that significantly enhanced the yield of soluble and functional protein. We demonstrate high-yield recovery of a native, high-molecular-weight RNA binding protein without the aid of fusion protein sequence. The principle factor that increased protein yield was the induction of protein expression in a late log phase culture, although reduced temperature during the induction and a low IPTG concentration also contributed to a higher yield.     

Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein.

We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alphaI/betaB loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent alphabeta loop of the C-domain to the same amino acid substitutions (MalE219). Moving the loop mutation from the N- to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super-secondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.     

Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations

The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time-dependent decrease in specific productivity. The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K(+) concentration. The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells. By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated.     

Exploitation of GFP fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins

A C-terminal green fluorescent protein (GFP) fusion to a model target protein, Escherichia coli CheY, was exploited both as a reporter of the accumulation of soluble recombinant protein, and to develop a generic approach to optimize protein yields. The rapid accumulation of CheY∷GFP expressed from a pET20 vector under the control of an isopropyl-β- d -thiogalactoside (IPTG)-inducible T7 RNA polymerase resulted not only in the well-documented growth arrest but also loss of culturability and overgrowth of the productive population using plasmid-deficient bacteria. The highest yields of soluble CheY∷GFP as judged from the fluorescence levels were achieved using very low concentrations of IPTG, which avoid growth arrest and loss of culturability postinduction. Optimal product yields were obtained with 8 μM IPTG, a concentration so low that insufficient T7 RNA polymerase accumulated to be detectable by Western blot analysis. The improved protocol was shown to be suitable for process scale-up and intensification. It is also applicable to the accumulation of an untagged heterologous protein, cytochrome c₂ from Neisseria gonorrhoeae , which requires both secretion and extensive post-translational modification.     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 611-622, 1997.     

Effect of operating variables on the yield of recombinant trypsinogen for a pulse-fed dilution-refolding reactor.

The inclusion body process route for manufacturing proteins offers distinct process advantages in terms of expression levels and the ease of initial inclusion body recovery. The efficiency of the refolding unit operation, however, does determine the overall economic feasibility of a process. Dilution refolding is the simplest and most extensively used refolding operation, although significant yield losses often occur due mainly to aggregation. Operating variables may have a significant effect on the degree of aggregation, but a systematic study has not been reported. This study investigates the effect of operating variables on the dilution refolding of solubilized r-trypsinogen inclusion bodies in a pulse-fed stirred reactor. Variables investigated were inclusion body washing, stirring speed, feed rate, concentration of solubilized r-trypsinogen, and concentration of urea during solubilization of the inclusion bodies. Additionally, the effect of baffles in the reactor was investigated. The yield of renatured r-trypsinogen varied between 12 +/- 0.2% and 21 +/- 1.0% depending on the specific combination of operating variables employed. It is clear that a suboptimal operating strategy can significantly reduce protein yield. In particular, we note that an increased intensity of mixing adversely affected yield in contrast to previous reports indicating that enhanced dispersion increases yield. We conclude that yield is determined not only by the efficiency of dispersion, but also by the local chemical environment of the protein as it folds, and the rate of change of this environment. This will be controlled by micromixing effects, and hence the intensity of agitation, in a complex manner requiring further characterization.     

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

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-βgalactosidase (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.     

Crystal structure of a defective folding protein

Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.     

Effect of sodium glycocholate and polyoxyethylene-9-lauryl ether on the hydrolysis of varying concentrations of insulin in the nasal homogenates of the albino rabbit

Protease inhibition has been postulated to be one of the several mechanisms by which penetration enhancers promote the mucosal absorption of peptide and protein drugs. The objective of this study was to determine whether protease inhibition by Na glycocholate and polyoxyethylene-9-lauryl ether, two extensively studied enhancers, led to suppression of insulin proteolysis over a range of insulin concentrations. To this end, the rate of insulin proteolysis in nasal tissue supernatants of the albino rabbit was determined in the presence of 0.1-2% Na glycocholate and polyoxyethylene-9-lauryl ether and at insulin concentrations ranging from 5 to 100 microM. Partly due to self-association, insulin was self-stabilizing against nasal proteolysis as its concentration was raised from 5 to 100 microM. At insulin concentrations lower than 50 microM, both Na glycocholate and polyoxyethylene-9-lauryl ether reduced the rate of insulin proteolysis. By contrast, at 100 microM insulin concentration, both enhancers accelerated insulin proteolysis. Such an effect was attributed to the deaggregation of insulin by the enhancers, increasing the proportion of monomers available for nasal proteolysis. The incorporation of 0.1 mM PCMPS, a potent inhibitor of insulin proteolysis, partly overcame the accelerating effect of Na glycocholate on insulin proteolysis.     

Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris

Simple cyclic fed-batch culture (cfbc), consisting of a constant medium feed with periodic withdrawals of culture, resulted in a product yield (13.4 mg protein per gram biomass) similar to that obtained using the complex multiphase industrial production strategy (13.7 mg protein per gram biomass). In cfbc, productivity was ultimately limited by the rate at which the cells could assimilate methanol. Glycerol was inhibitory to growth at high concentrations. However, product yield continued to increase as the glycerol concentration was increased. In chemostat culture, dissolved oxygen concentration influenced product yield independently of any detectable influence on cell growth.     

Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli

The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

The <Emphasis Type="Italic">E. coli</Emphasis> pET expression system revisited—mechanistic correlation between glucose and lactose uptake

Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q _s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q _s,lac) and q _s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q _s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q _s,lac and q _s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.