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Over-expression and single-step purification of human IFNα8 and human IFNα2b reveals the highest antiviral activity of human IFNα8
Microbial Cell Factories volume 5, Article number: P73 (2006)
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 .
Comparing the rare codon compositions of both HuIFNα subtypes, two clusters of 30 amino acids, containing five minor triplets, were found (see Figure 1). For the HuIFNα2b gene, the minor codons, represented only by AGG/AGA (that encodes for Arg) are present in both clusters. However, besides the minor triplets that encodes for Arg, in the HuIFNα8 gene one minor codon AUA (ATA triplet, that encodes for Ile) is present in the first cluster, while in the second one, the CUA (CTA triplet, that encodes for Leu) exists.
Taking into account the minor codon composition in both HuIFNα genes, E. coli BL21-codonplus-RIL cells (Stratagene, USA) were used to increase the heterologous polypeptides expression levels. E. coli BL21-codonplus-RIL cells were transformed with the plasmids pALF8-4 and pAGUA-4  coding for HuIFNα8 and HuIFNα2b, respectively. This strain carries out additional copies of the dna Y, ile Y and leu W tRNAs in an additional Cmr pACYC derivative plasmid. The last two tRNA decode the AUA and CUA minor codons, respectively. The obtained transformants were grown in M9 minimum medium. For HuIFNα8 transformants, the protein expression level increased from 1% (with E. coli BL-21/pALF8-4) up to 25% (with E. coli BL-21 CP RIL/pALF8-4) of total bacterial proteins based on densitometric analysis of scanned gels (see Figure 2). Similar results were obtained with the HuIFNα2b Ampr-Cmr transformants, the expression levels increased from 5% (with E. coli BL-21/pAGUA-4) up to around 20% (with E. coli BL-21CPRIL/pAGUA-4) of total host proteins.
The purity of purified HuIFNα subtypes was estimated over 98%, based on the homogeneity observed in the silver stained SDS-PAGE and by RP-HPLC analysis (see Figure 3).
The antiviral potency of both HuIFNα subtypes was compared in an in vitro assay with one microgram of each protein challenged with mengo virus . Results of five independent experiments are shown in the table 1. The HuIFNα8 had 1,46 fold more antiviral activity than HuIFNα2b (p < 0,05) in this biological system.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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García, J.C.S., Ariza, A.M., Lassa, A.M. et al. Over-expression and single-step purification of human IFNα8 and human IFNα2b reveals the highest antiviral activity of human IFNα8. Microb Cell Fact 5, P73 (2006). https://doi.org/10.1186/1475-2859-5-S1-P73
- Hairy Cell Leukemia
- Rare Codon
- Codon Composition
- High Antiviral Activity
- Human Alpha Interferon