Over-expression and single-step purification of human IFN_α8 and human IFN_α2b reveals the highest antiviral activity of human IFN_α8

Background

Human alpha interferons (_HuIFN_α) comprise a multigene family, originally identified as proteins responsible for the induction of cellular resistance to viral infections, subdivided into 13 different subtypes (IFN_{α1, 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 IFN_α subtypes [2, 3] and it was found that the activity varied greatly depending on the target cells, the IFN_α subtypes and even if the proteins were compared on base of both units of biological activity or mass [4]. The _HuIFN_α2b and _HuIFN_α8 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 _HuIFN_α8 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 _HuIFN_α 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 _HuIFN_α8 in E. coli without success [13]. In the present work, the heterologous expression of both _HuIFN_α8 and _HuIFN_α2b 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 _HuIFN_α 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 _HuIFN_α subtypes, two clusters of 30 amino acids, containing five minor triplets, were found (see Figure 1). For the _HuIFN_α2b 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 _HuIFN_α8 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

Clusters of 30 aminoacid presents in both_HuIFN_α 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 _HuIFN_α 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 _HuIFN_α8 and _HuIFN_α2b, respectively. This strain carries out additional copies of the dna Y, ile Y and leu W _tRNAs in an additional Cm^r 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 _HuIFN_α8 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 _HuIFN_α2b Amp^r-Cm^r 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

Expression of both _HuIFN_α 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 _HUIFN_α2 and _HUIFN_α8. 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 _HuIFN_α 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

(A). RP-HPLC analysis of purified _HUIFN_α2 (A) and _HUIFN_α8 (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 _HuIFN_α8 and _HuIFN_α2b are correctly linked by disulfide bridges ((Data not show).

The antiviral potency of both _HuIFN_α 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 _HuIFN_α8 had 1,46 fold more antiviral activity than _HuIFN_α2b (p < 0,05) in this biological system.

Table 1 Antiviral potency of both purified _HuIFN_α subtypes.

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 _HuIFN_α8 shown in vitro add new evidences to take into account this proteins as strong therapeutic candidate.