Recombinant Consensus Interferon Research Articles

Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN.

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14g/L) in shake flask experiment resulted in higher yield as compared with 1mM IPTG. Finally, with single-step purification on DEAE sepharose, 150mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.

Optimization of conditions for high-level expression and purification of human recombinant consensus interferon (rh-cIFN) and its characterization.

Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5kDa and specific activity was 2.0 × 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.

Use of Recombinant Adenovirus Vectored Consensus IFN-α to Avert Severe Arenavirus Infection

Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

Cloning recombinant consensus interferon with overlap extension polymerase chain reaction

Cloning recombinant consensus interferon with overlap extension polymerase chain reaction

Identification of an oxidative refolding intermediate of recombinant consensus interferon from inclusion bodies and design of a two‐stage strategy to promote correct disulfide‐bond formation

Dilution refolding of recombinant consensus IFN (interferon) from inclusion bodies suffers from low yield. A stable intermediate was found to mix with the correct product and to have an antiviral activity of less than 10% of the latter. This intermediate would form precipitates upon removal of the precipitation inhibitor arginine. Compared with the native protein, the intermediate moved more slowly on non-reducing SDS/PAGE. The CD and fluorescence spectra indicated that it had formed a native-like structure, but had only one disulfide bond: Cys(29)-Cys(139). Further evidence showed that the formation of Cys(29)-Cys(139) is specific and very likely to happen, even in the presence of a high concentration of reducing agent, whereas pairing of the other disulfide (Cys(1)-Cys(99)) needed a stronger oxidative condition. It competed with intermolecular disulfide bonding to form covalent oligomers. On the basis of this discovery, a two-stage refolding step strategy was designed that employed a modified dilution refolding step followed by a dialysis refolding step. The first stage used a high concentration of reducing agent together with the precipitation inhibitor arginine. The purpose was to hinder any reaction through Cys(1) or Cys(99) but allow the intramolecular disulfide bonding of Cys(29)-Cys(139). The second stage was a dialysis step that gradually increased the oxidative agent concentration and simultaneously decreased the arginine concentration. The refolding yield was increased from 35 to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed.

A newly proposed mechanism for arginine-assisted protein refolding—not inhibiting soluble oligomers although promoting a correct structure

Arginine has been demonstrated to be capable of suppressing aggregation during protein refolding. However, the pathway and the mechanism for arginine to participate in and to assist refolding process still remains unclear. In this study, arginine-assisted refolding of recombinant consensus interferon (rIFN-con 1) was investigated. It was found that although arginine minimized the formation of protein precipitate, it failed to prevent the formation of the soluble oligomeric species. The amount of the oligomers increased with the increase in arginine concentration. This phenomenon has not been reported. On the other hand, arginine was able to promote the yield of correctly refolded rIFN-con 1, which was more than 2 times higher than that in the absence of arginine. A proposed mechanism is the stabilization of different soluble species by arginine, which slowed down the conformational movement. The stabilization effect on native-like structure formation overwhelmed the oligomeric promotion effect, which resulted in a composite effect of increased refolding yield for rIFN-con 1 when arginine concentration was below 0.5 M.

Comparison of an antiviral activity of recombinant consensus interferon with recombinant interferon-α-2b

To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-α-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-α-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.

Stability of Recombinant Consensus Interferon to Air‐Jet and Ultrasonic Nebulization

The stability of recombinant consensus α‐interferon (rConIFN) to air‐jet and ultrasonic nebulization was evaluated. Volumes of 10 mL of 0.5mg/mL rConIFN in phosphate‐buffered saline (PBS) at pH 6.3 were nebulized with a Collison three‐jet nebulizer at 40 psig (10 L/min) for up to 25 min. The effects of pH (3.0, 6.3, and 9.0), additive (0.1% w/v Tween 80, 0.1% w/v Tween 20, and 1% w/v PEG 8000), and ionic strength (0, 0.25, and 1.0) were examined. The effects of ultrasonic nebulization were studied using three devices (DeVilbiss “Aerosonic”; Mountain Medical “Microstat”, and Medix “Easimist”). Stability of rConIFN was assessed by size exclusion chromatography and native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Structural effects were examined by circular dichroism spectroscopy and bioactivity was assessed by anin vitromitogenic inhibition bioassay. rConIFN is destabilized by air‐jet nebulization. Insoluble noncovalent aggregates are produced rapidly, and only ≈25% of the initial monomeric protein remains after 25 min of nebulization. This correlates with a decrease inin vitrobioactivity. Aggregation during nebulization is influenced by pH (9.0<6.3<3.0) but even at the highest pH, >25% aggregation is observed. Ionic strength does not appear to influence aggregation. rConIFN is also seen to adhere to glass after nebulization. Samples from a rinse of the emptied reservoir with 0.1% w/v SDS, after thorough rinsing with water (three times), show a strong rConIFN band on SDS–PAGE gels. The use of PEG 8000 and Tween mitigate aggregate formation and adhesion (<20%). The cumulative output collected as a wet or dry aerosol is not aggregated to the same extent as the residual protein remaining in the nebulizer. Ultrasonic nebulization also results in aggregation, but the extent of denaturation is dependent upon the nebulizer used and is related to the heating of nebulizer solutions. Cooling of the nebulizer solution during operation (<30 °C) minimizes aggregation (<5%), and bioactivity is retained.

Systemic absorption and activity of recombinant consensus interferons after intratracheal instillation and aerosol administration.

The pulmonary pharmacokinetics and bioactivity of E. coli derived recombinant consensus interferon (CIFN) and a modified lactose-conjugated consensus interferon (LacCIFN) were evaluated in animals. Estimated doses of 20 and 100 micrograms/kg of the interferons were administered to anesthetized rats by aerosol via ultrasonic nebulizer as well as intravenous injection. Rats also received nominal doses of 50 and 200 micrograms/kg via intratracheal instillation (IT). Hamsters were treated with interferon via various routes including IT. The effectiveness of treatment was assessed by the resistance to development of hind leg paralysis following infection with encephalomyocarditis virus. Significant amounts of CIFN and LacCIFN were found in rat plasma after aerosol administration. Peak plasma levels occurred approximately 25-30 minutes with estimated bioavailabilities approaching 70%. Absorption of CIFN was rate limiting and plasma levels were detectable 12 hr post-dose. The CIFN at IT doses as low as 5 micrograms/kg was effective at reducing paralysis in hamsters but protection was variable and doses of up to 100 micrograms/kg were not 100% effective. Despite the incomplete protection, the results demonstrate the feasibility of treating systemic viral infections with interferon administered directly to the lungs.