- Poster Presentation
- Open Access
Over-expression and single-step purification of human IFNα8 and human IFNα2b reveals the highest antiviral activity of human IFNα8
© García et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Hairy Cell Leukemia
- Rare Codon
- Codon Composition
- High Antiviral Activity
- Human Alpha Interferon
Human alpha interferons (HuIFNα) comprise a multigene family, originally identified as proteins responsible for the induction of cellular resistance to viral infections, subdivided into 13 different subtypes (IFNα1, α2, α4, α5, α6, α7, α8, α10, α13, α14, α16, α17 and α21). The genes that encode these proteins are clustered on chromosome 9 in human . There have been reports of obvious differences in the relative biologicals activities among IFNα subtypes [2, 3] and it was found that the activity varied greatly depending on the target cells, the IFNα subtypes and even if the proteins were compared on base of both units of biological activity or mass . The HuIFNα2b and HuIFNα8 are among the IFN subtypes with highest antiviral activity. The first one was licensed by the Food and Drug Administration in 1986 (USA) for the treatment of hairy cell leukemia . On the other hand, the HuIFNα8 has shown the highest biological activity in several in vitro assays [6–8].
Several progresses in the fundamental understanding of transcription, translation, and protein folding in Escherichia coli, together with the availability of improved genetic tools (including new mutants) are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins. Several strategies have been required to enhance rare codons gene expression [9, 10].
Homogenous HuIFNα preparations were originally obtained in 1978 for further chemical and physical characterization . Thereafter have been introduced new high performance chromatography techniques useful to obtain enough quantity of these proteins for their chemical, biological and immunological studies . Although high quantities of these proteins could be obtained, these procedures involve costly and laborious purification protocols.
In our previous report we attempted to over express the HuIFNα8 in E. coli without success . In the present work, the heterologous expression of both HuIFNα8 and HuIFNα2b was significantly improved. These proteins were purified by one step and low cost SDS-PAGE procedure and used for detailed chemical and physic characterization. Both HuIFNα subtypes obtained were used to compare its antiviral activity by using an in vitro model involving Hep-2 cells challenged with Mengo virus .
Antiviral potency of both purified HuIFNα subtypes.
E. coli BL-21 CP RIL
In summary, this work highlights several issues to obtain human proteins with high molecular homogeneity avoiding costly and tedious purification procedures. Therefore, we recommend this experimental strategy to obtain humans proteins with high therapeutic potentials. The highest antiviral activity of HuIFNα8 shown in vitro add new evidences to take into account this proteins as strong therapeutic candidate.
The authors thank Prof. Jose Luis Fernandez Sierra for critical reading of the manuscript, and especially to Prof. Magaly Garcia Blanco for her substantial contribution and support to this work.
- Pestka S, Krause CD, Walter MR: Interferons, interferon-like cytokines, and their receptors. Immunol Rev. 2004, 202: 8-32. 10.1111/j.0105-2896.2004.00204.x.View ArticleGoogle Scholar
- Foster GR, Rodrigues O, Ghouze F, Schulte Frohlinde-E, Testa D, Liao MJ, Stark GR, Leadbeater L, Thomas HC: Different relative activities of human cell-derived interferon-alpha subtypes: IFN-alpha 8 has very high antiviral potency. J Interferon Cytokine Res. 1996, 16: 1027-1033.Google Scholar
- Yanai Y, Sanou O, Kayano T, Ariyasu H, Yamamoto K, Yamauchi H, Ikegami H, Kurimoto M: Analysis of the antiviral activities of natural IFN-alpha preparations and their subtype compositions. J Interferon Cytokine Res. 2001, 21: 835-841. 10.1089/107999001753238088.View ArticleGoogle Scholar
- Blatt L, Davis J, Klein S, Taylor M: The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon. J Interferon Cytokine Res. 1996, 16: 489-499.View ArticleGoogle Scholar
- Bekisz J, Schmeisser H, Hernandez J, Goldman N, Zoon K: Human interferons alpha, beta and omega. Growth Factors. 2004, 22: 243-251. 10.1080/08977190400000833.View ArticleGoogle Scholar
- Yamamoto S, Yano H, Sanou O, Ikegami H, Kurimoto M, Kojiro M: Different antiviral activities of IFN-alpha subtypes in human liver cell lines: synergism between IFN-alpha2 and IFN-alpha8. Hepatol Res. 2002, 24: 99-115. 10.1016/S1386-6346(02)00020-7.View ArticleGoogle Scholar
- Foster G, Rodrigues O, Ghouze F, Schulte Frohlinde-E, Testa D, Liao M, Stark G, Leadbeater L, Thomas H: Different relative activities of human cell-derived interferon-alpha subtypes: IFN-alpha 8 has very high antiviral potency. J Interferon Cytokine Res. 1996, 16: 1027-1033.Google Scholar
- Yanai Y, Horie S, Yamamoto K, Yamauchi H, Ikegami H, Kurimoto M, Kitamura T: Characterization of the antitumor activities of IFN-alpha8 on renal cell carcinoma cells in vitro. J Interferon Cytokine Res. 2001, 21: 1129-1136. 10.1089/107999001317205268.View ArticleGoogle Scholar
- Foster G, Masri S, David R, Jones M, Datta A, Lombardi G, Runkell L, Sizing I, James M, Marelli Berg-F: IFN-alpha subtypes differentially affect human T cell motility. J Immunol. 2004, 173: 1663-1670.View ArticleGoogle Scholar
- Zhou Z, Schnake P, Xiao L, Lal A: Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization. Protein Expr Purif. 2004, 34: 87-94. 10.1016/j.pep.2003.11.006.View ArticleGoogle Scholar
- Rubinstein M, Rubinstein S, Familletti PC, Gross MS, Miller RS, Waldman AA, Pestka S: Human leukocyte interferon purified to homogeneity. Science. 1978, 202: 1289-1290. 10.1126/science.725605.View ArticleGoogle Scholar
- Pestka S: The human interferon alpha species and receptors. Biopolymers. 2000, 55: 254-287. 10.1002/1097-0282(2000)55:4<254::AID-BIP1001>3.0.CO;2-1.View ArticleGoogle Scholar
- Acosta Rivero-N, Sanchez JC, Morales J: Improvement of human interferon HUIFNalpha2 and HCV core protein expression levels in Escherichia coli but not of HUIFNalpha8 by using the tRNA(AGA/AGG). Biochem Biophys Res Commun. 2002, 296: 1303-1309. 10.1016/S0006-291X(02)02056-9.View ArticleGoogle Scholar
- Santana H, Martínez E, Sánchez JC, Moya G, Sosa A, Hardi E, Beldarrain A, González LJ, Betancourt L, Besada V, Currás T, Ferrero J, Pujols V, Gil M, Herrera L: Molecular characterization of recombinant human interferon alpha-2b produced in Cuba. Biotecnologia Aplicada. 1999, 16: 154-159.Google Scholar
This article is published under license to BioMed Central Ltd.