Recombinant Human Interferon Research Articles

Proteome analysis of Escherichia coli periplasmic proteins in response to over-expression of recombinant human interferon α2b

The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins.

Evaluation of Neuroimmunomodulatory Activity of Recombinant Human Interferon α

Objective: Recombinant human interferon (rhIFN)-α is a potent immunoregulator having a wide range of therapeutic applications. In the present study, rhIFN-α was evaluated for its neuroimmunomodulatory activity. Method: Dose-dependent gene expression of cytokines and chemokines in the brain of rhIFN-administered mice was studied using real-time SYBR green PCR. Results: Statistically significant increase in expression levels of tumor necrosis factor-α, interleukin (IL)-6, IL-1β and IFN-γ were observed. Conclusion: The findings indicate that rhIFN-α may be used at an optimized dose to cause appropriate neuromodulation of cytokine/chemokine secretion that can aid in the development of therapeutic approaches for many infectious diseases of the central nervous system for which therapies are lacking.

Metabolic changes of recombinant Escherichia coli BL21 (DE3) during overexpression of recombinant human interferon beta in HCDC

The influence of the overexpression of recombinant human interferon beta (rhINF-β) during high cell density cultivation (HCDC) on metabolic characteristics of recombinant E. coli BL21 (DE3) was studied. Fed-batch culture technique was employed to grow the host cell for the production of human interferon beta in pET expression system in a 5L bench-top bioreactor. The final cell density of 42.15 g L-1 and cell growth (OD600nm) of 103.5 were obtained respectively in fed-batch culture experiment. Four hours after the induction, the specific growth rate was decreased. The initial concentration of the glucose, ammonium and phosphate in the TB culture medium were 5.0, 12.0 and 3.12 g L-1 respectively. The maximum amount of the produced rhINF-β was 2.8 g L-1 after applying the fed-batch cultivation technique. Besides, time profiles of the acetate and lactate production were similar, the acetate concentration (4.91 g L -1 ) was lower than that of lactate (10.08 g L -1 ) and the

Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-α, shows antitumor effect in vitro and in vivo

ObjectiveA recombinant antitumor/antiviral protein (Novaferon, Nova) is a new type of interferon, which is produced by artificial design technology combining DNA-shuffling and High Throughput Screening (HTS).MethodsThe in vitro biological activities, such as anti-tumor activity and antiviral activity of Nova and recombinant human interferon alpha-2b (rhIFN-α2b) was performed; in vivo anti-tumor activity in nude mice was also tested. Flow cytometry, histo-pathological analysis including HE staining and immunohistochemistry, and surface plasmon resonance assay were performed to investigate the underlying mechanisms analysis.ResultsNova exhibited stronger anti-cancer effects compared to rhIFN-α2b in vitro and in vivo. The antitumor mechanisms of Nova may be related to S phase arrest, pro-apoptosis, and inhibition of tumor angiogenesis. Moreover, Nova exhibited a higher binding affinity for IFN receptor 2 (IFNR2) than rhIFN-α2b, which is one of the possible reasons accounting for its stronger actions against tumor cells compared with rhIFN-α2b.ConclusionNova has strong antitumor activity and could be a potentially effective therapeutic drug for cancer.

Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli.

Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins.

Study on the effect of solution conditions on heat induced-aggregation of human alpha interferon.

A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b (rhIFNα2b) has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFNα2b. The protein was incubated for 1 h at 40-70 °C and for up to 240 h at 50 °C and its aggregation tendency was then studied using optical density (at 350 nm), SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH (5, 6 and 7) and buffer concentrations (10, 55 and 100 mM) on the aggregation of protein following incubation at 50 °C for 72 h was also evaluated. The results obtained for samples incubated at 50 °C for up to 240 h showed that OD350 and the amount of higher molecular weight aggregates (HMW) increased and the monomer content decreased significantly (p<0.05) as the incubation time increased. Following incubation at various temperatures, a significant increase in OD350, drop in monomer content and increase in the amount of HMW aggregates were observed (p<0.05). Data obtained from incubation of samples at 50 °C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 (p<0.05). At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFNα2b.

Studies to analyse the relationship between IFNα2b gene dosage and its expression, using a Pichia pastoris‐based expression system

Human interferon α2b (hIFNα2b) is the most important member of the interferon family. Escherichia coli, yeasts, mammalian cell cultures and baculovirus-infected insect cells have been used for expressing recombinant human interferon. Recently a Pichia pastoris-based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered an important factor in obtaining the optimum expression of recombinant protein, which may vary from one protein to another. In the present study we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b-expressing cassettes were created via an in vitro multimerization approach. P. pastoris host strain X-33 was transformed using these expression cassettes. Groups of P. pastoris clones transformed with different copies of the IFNα2b expression cassette were screened for intrachromosomal integration. The IFNα2b expression level of stable transformants was checked. The copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that an increase in copy number generally had a positive effect on the expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. It was also observed that an increase in drug resistance of a clone did not guarantee its high expression, as integration of a marker gene did not always correlate with integration of the gene of interest.

Multiple microvenular hemangioma: A case report

The current study reports a case of multiple microvenular hemangioma (MH). A 35-year-old male presented with dark red maculopapules on the trunk and limbs that had been apparent for 5 years. The number of lesions exceeded 100 in total. A histological examination demonstrated multiple, irregular, branching venules in the dermis, without any endothelial atypia. On immunohistochemical analysis of the vascular structures, the endothelial cells stained positive for CD31, CD34 and factor VIII, and the perivascular cells stained positive for SMA and HHF-35. These observations were consistent with a diagnosis of MH, and should be differentiated from the most common differential diagnosis of patch-stage Kaposi’s sarcoma. There was no clear effect following topical application of recombinant human interferon α-2b gel.

Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains

The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis.

Photodynamic therapy combined with recombinant human interferon α-2b gel for the treatment of male multiple anorectal condyloma acuminatum:a clinical observation

Objective To investigate the clinical efficacy and safety of 5-aminolaevulinic acid-photodynamic therapy(ALA-PDT) in treatment of anorectal condyloma acuminatum.Methods 95 cases who suffered from male multiple condyloma acuminatum were randomly divided into two groups.There were 50 cases in the treatment group and 45 cases in the control group.There were no statistically significant between two groups in age,sex,course of disease,skin lesions,the size and the distribution.The treatment group was treated by photodynamic therapy combined with recombinant human interferon α-2b Gel while the control group was treated only by photodynamic therapy.The treatment time lasted 7-10 days later if the Lesion was not completely removed.Totally,1-4 treatment sessions were given.Then the rate of recovery and recurernce of the two groups were compared.Results The recovery rate in treatment group was 94.0％ (47/50) and 88.9％ (40/45) were found in control group.There were no significant difference in the two groups (x2 =0.290,P ＞ 0.05) ;The recurernce rate of the treatment group was 2.0％ (1/50) and the control group was 13.3％ (6/45),the two groups had significant differences (x2 =4.457,P =0.035).Conclusion ALA-PDT combined with recombinant human interferon α-2b Gel in the topical treatment of anorectal condyloma acuminatum is safe and effective,whcih may serve a therapeutic option for anorectal condyloma acuminatum. Key words: Condylomata acuminata; Aminolaevulinic acid; Recombinant human interferon α-2b

Improved detection of variants in recombinant human interferon alpha-2a products by reverse-phase high-performance liquid chromatography on a core–shell stationary phase

The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core–shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.

Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b

Interferon alpha 2b (IFNα-2b) is an important cytokine and used for antiviral and anticancer treatment. The low cost production of IFNα-2b with high biological activity is necessary to provide the interferon therapy to the hepatitis patients in Pakistan. In the present study, human interferon alpha 2b (hIFNα-2b) gene from a healthy person was cloned and overexpressed in E. coli BL21(DE3). The molecular weight of the expressed hIFNα-2b is 19 kDa. The over expressed recombinant hIFNα-2b was checked by ELISA using antibodies raised against commercially available hIFNα-2b. The biocomputational analysis of recombinant hIFNα-2b gene showed the 99.9% nucleotide sequence and 100% deduced amino acid sequence homology with reported sequences of IFNα-2b. The predicted 3D-structure showed mainly five α-helices, one 310 helix and two disulfide bonds at Cys1-Cys98 and Cys129-Cys138. The amino acid sequence alignment indicated that the disulfide linkage position is conserved in all IFNα family members. On the basis of sequence homology among interferon alpha family, new potent variants of hIFNα-2b with enhance efficacy can be produced. Indigenous production of IFNα-2b from gene of local population will reduce the cost and increase tolerability of interferon therapy.

Effect of combining loop electrosurgical excision procedure and recombinant human interferon α2b suppository in treatment of cervical intraepithelial neoplasia

Objective To study the effect of combining loop electrosurgical excision procedure (LEEP) and recombinant human interferon α2b (rhIFNα-2b) suppository in treatment of cervical intraepithelial neoplasia (CIN).Methods Prospective,random and control study was conducted in 82 patients with CINⅠ-CINⅢ.Before canting out LEEP to these patients,all women were performed HPV DNA detection by the method of Hybri Max.Among these patients,41 patients were assigned to the studying group,in which the patients were given rhIFNα-2b suppository for three courses of treatment after LEEP.The other 41 patients who carried out LEEP simply were assigned to the control group.Liquid-based ThinPrep cytology test (TCT) and HPV DNA were examined in the sixth and twelfth month after treatment.Results In the studying group,the cure rate was 90.2％ when LEEP ended six months,and the cure rate was 100.0％ when LEEP ended twelve months.In the control group,the cure rate was 43.9％ when LEEP ended six months,and the cure rate was 61.0％ when LEEP ended twelve months.In the sixth and twelfth month after LEEP,the difference was significant when we compared the cure rate between the two groups (x2 =19.93,19.89,all P ＜ 0.05).Conclusion The clinical effect of combining LEEP and rhIFNα-2b suppository is better than LEEP in treatment of CIN.The method can remove or destroy the cervical lesions effectively and inhibit HPV replication and spread of HPV infection. Key words: Loop electrosurgical excision procedure; Interferon alfa-2b suppository; Cervical intraepithelial neoplasia; Human papillomavirus

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.

The ETR (End Treatment Response) of Chinese Brand Human recombinant interferon alpha 2a plus Ribavirin in chronic hepatitis C patients selected from Combined Military Hospitals in Pakistan

This Study was carried out to determine the End of Treatment Response (ETR) of a Chinese Source Human Recombinant Interferon Alpha 2a and Ribavirin in chronic hepatitis C affected patients, selected from different combined military hospitals in Pakistan. In this study hepatitis affected noncirrhotic patients from different combined military hospitals in Pakistan were selected from September 2010 to April 2011. Sixty seven patients were selected based on the inclusion exclusion criteria. Fifty seven patients completed the 24 weeks therapy for Hepatitis C. The Patients (n=57) had persistently raised serum aminotransferase (ALT), positive HCV antibodies by 3rd generation ELISA, positive HCV RNA by polymerase chain reaction. Study group patients underwent 24 weeks therapy with a Chinese source Interferon and Ribavirin and subsequently followed up for the End of Treatment Response by the assay of HCV RNA by polymerase chain reaction at 24 weeks. Patients of chronic hepatitis C (44 males and 13 females) had age range 20-56 years. After 24 weeks of therapy with a Chinese source Interferon and Ribavirin, 73.7% patients showed a favourable ETR manifested by Negative HCV RNA polymerase chain reaction.Treatment with a Chinese source Interferon and Ribavirin combination therapy for 24 weeks produces a favourable End of Treatment Response in patients of chronic hepatitis C from different military hospitals in Pakistan.

Refolding of recombinant human interferon gamma inclusion bodies in vitro assisted by colloidal thermo-sensitive poly(N-isopropylacrylamide) brushes grafted onto the surface of uniform polystyrene cores

Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γin vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55nm and 110nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33°C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15°C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro.

Characterization of physiochemical and biological properties of spherical protein crystals for sustained release

This study presents an exploration on extending the action of therapeutic proteins by crystallization strategy without new molecular entities generation. Recombinant human interferon α-2b (rhIFN), a model protein drug in this case, was crystallized using a hanging drop vapor diffusion method. A novel chelating technique with metal ions was employed to promote crystals formation. The physico-chemical characterization of the protein crystals, including morphology, particle size, X-ray diffraction, circular dichroism and biological potency evaluations were performed. In addition, the in vitro release behavior of rhIFN from crystal lattice suggested an exciting possibility of protein crystals as a long-acting formulation. The work described here demonstrates the possibility of spherical crystals of biomacromolecules for controllable delivery application of therapeutic proteins.

Effects of topical ocular administration of high doses of human recombinant interferon alpha-2b and feline recombinant interferon omega on naturally occurring viral keratoconjunctivitis in cats

To determine whether 14-day topical ocular administration of high doses of feline recombinant interferon omega (FelFN) or human recombinant interferon alpha-2b (HulFN) solution improves clinical disease and decreases virus shedding in cats with naturally acquired viral keratoconjunctivitis. 36 cats with upper respiratory tract disease and ocular involvement. Cats received 1 drop of FelFN solution (1 × 10(6) U/mL), HulFN solution (1 × 10(6) U/mL), or saline (0.9% NaCl) solution (12 cats/group) in each eye twice daily for 14 days (beginning day 1). Oropharyngeal and conjunctival swab samples were collected from each cat before (day 0) and on day 14 of treatment for virus isolation (VI) and real-time quantitative PCR (RT-qPCR) testing to detect feline herpesvirus-1 and feline calicivirus. Subjective clinical scores were recorded on days 0, 3, 7, 10, and 14. The number of cats for which feline herpesvirus-1 was detected via VI or RT-qPCR assay was generally (albeit not always significantly) lower on day 14, compared with day 0 findings; however, findings on days 0 or 14 did not differ among groups. The number of cats for which feline calicivirus was detected via VI or RT-qPCR assay did not differ significantly between days 0 and 14 for any group. Clinical scores significantly decreased over the 14-day period but did not differ among groups. In cats with naturally occurring viral keratoconjunctivitis, bilateral ocular administration of high doses of FelFN or HulFN twice daily for 14 days did not improve clinical disease or virus shedding, compared with treatment with saline solution.

Evaluation of Adverse Effects of Mutein Forms of Recombinant Human Interferon Alpha-2b in Female Swiss Webster Mice

Purpose. We successfully developed recombinant human interferon alpha-2b (rhIFN-α2b) and mutein forms through the site-directed mutagenesis technique. The mutein forms were developed by substituting cysteins at positions 2 and 99 with aspartic acids. The potential adverse effects of these rhIFN-α2bs were assessed by acute and subchronic studies. Methods. In the acute study, rhIFN-α2bs were subcutaneously administered to mice at a single dose of 97.5 μg/kg, 975 μg/kg, and 9.75 mg/kg BW and were observed for 14 days. In the subchronic study, single dose of 1.95 μg/kg and 19.5 μg/kg, respectively, was given subcutaneously every 3 days for 45 days. Results. No death as well as abnormality in body weight, behavior, presentation of main organs, and value of plasma SGPT and SGOT was observed. Wild type and mutein rhIFN-α2bs did not show significant adverse effects at dose up to 9.75 mg/kg BW. Administration of these rhIFN-α2bs given repeatedly did not induce any adverse effect. Conclusion. These results suggest that our rhIFN-α2bs are safe. However, further study is still needed to clarify the safety issue before use in clinical trial.

Refolding of Recombinant Human Interferon α-2a fromEscherichia coliby Urea Gradient Size Exclusion Chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon alpha-2a (rhLFNalpha-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhLFNalpha-2a would pass along the 8.0-3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 x 10(8) IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhLFNalpha-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhl FNalpha-2a refolding in terms of specific activity and mass recovery.