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  • Open Access

Over-expression and single-step purification of human IFNα8 and human IFNα2b reveals the highest antiviral activity of human IFNα8

  • 1,
  • 1,
  • 2,
  • 2 and
  • 2
Microbial Cell Factories20065 (Suppl 1) :P73

https://doi.org/10.1186/1475-2859-5-S1-P73

  • Published:

Keywords

  • Hairy Cell Leukemia
  • Rare Codon
  • Codon Composition
  • High Antiviral Activity
  • Human Alpha Interferon

Background

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 [1]. 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 [4]. 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 [5]. On the other hand, the HuIFNα8 has shown the highest biological activity in several in vitro assays [68].

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 [11]. Thereafter have been introduced new high performance chromatography techniques useful to obtain enough quantity of these proteins for their chemical, biological and immunological studies [12]. 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 [13]. 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 [14].

Results

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.
Figure 1
Figure 1

Clusters of 30 aminoacid presents in bothHuIFNα subtypes. In boxes, the AGA/AGG triplets coding for Arginine. The ATA and CTA triplets coding for Isoleucine and Leucine, respectively, are underlined.

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 [13] 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.
Figure 2
Figure 2

Expression of both HuIFNα subtypes in E. coli cells grown in minimum media. (A) 12.5% SDS-PAGE stained with Coomassie blue, and (B) Immunoblotting analysis using a mixture of rabbit polyclonal antibodies specific for both HUIFNα2 and HUIFNα8. Samples: (1) E. coli BL-21/ pALF8-4, (2) E. coli BL-21CP RIL/ pALF8-4, (3), E. coli BL-21CP RIL/ pAGUA-4, (4) E. coli BL-21/ pAGUA-4, (5) E. coli BL-21CP RIL. The molecular weight markers (M) are indicated (M).

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).
Figure 3
Figure 3

(A). RP-HPLC analysis of purified HUIFNα2 (A) and HUIFNα8 (B). The molecular weight of markers shown in SDS-PAGE are: 34 kDa, 26 kDa, 20 kDa and 7 kDa, respectively. (B). The LC-MS analysis of the tandem digestion tryptic/V8 revealed that cysteine residues of HuIFNα8 and HuIFNα2b are correctly linked by disulfide bridges ((Data not show).

The antiviral potency of both HuIFNα subtypes was compared in an in vitro assay with one microgram of each protein challenged with mengo virus [14]. 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.
Table 1

Antiviral potency of both purified HuIFNα subtypes.

Sample

Titer 1

Titer 2

Titer 3

Titer 4

Titer 5

Titer average

HuIFNa8

5.1

4.5

4.8

3.9

4.1

4.48 (3.86-5.09)a

HuIFNa2b

2.3

2.8

2.5

3

2.1

2.54 (2.08-2.99)a

E. coli BL-21 CP RIL

nd

nd

nd

nd

nd

nd

The titer values are expressed in 106 international units. nd, not antiviral activity detectable.

a Values in parenthesis correspond to confidence limits (p < 0.05).

Conclusion

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.

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Process Control Department, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P., 10600 Havana C., Cuba
(2)
Division of Physical-Chemistry, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P., 10600 Havana C., Cuba

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Copyright

© García et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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