Biological Activity of Optimized Codon Bovine Type III Interferon Expressed in Pichia pastoris

Abstract

Type III interferons (IFN-λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn-λ (boifn-λ) gene were synthetized after codon optimization. The boifn-λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN-λ3^V18M. The recombinant plasmid pPICZαA-boIFN-λ3/λ3^V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high-level extracellular soluble form. Dominant expression strains of boIFN-λ3/λ3^V18M were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN-λ3/λ3^V18M exceeded 10⁶ U/mg, and they were neutralized with IFN-λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN-λ3/λ3^V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 10⁴ U/mL. Overall, boIFN-λ3 and boIFN-λ3^V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN-λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN-λs and provide material for therapeutic development.

Keywords: Pichia pastoris; antiproliferative activity; antiviral activity; bovine interferon–λ3; codon optimization; glycosylation.

Figure 1

Construction of pPICZαA–boIFN–λ3 by SOE with optimized codons.

Figure 2

Sequence alignment of codon–optimized boIFN–λ3^V18M and original boIFN–λ3. (A). The translation process of IFN–λ3. (B). Sequence alignment of IFN–λ3. GenBank Accession: HuIFN–lambda3, NM_172139.2; MuIFN–lambda3, NM_177396.1; BoIFN–lambda3V18M, No. OQ565419; BoIFN–lambda3, No. OQ565418. Identical and similar residues are boxed in red and yellow, respectively. The residues representing the mutation site are within the indigo frame. (C). Optimization for codon usage rate of the bovine ifn–λ3 gene in P. pastoris. The percentage distribution of codons was computed in codon quality groups. The value of 100 was set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. Codons with values lower than 30 are likely to hamper expression efficiency. (D). Comparison of the values of index optimization. The possibility of a high protein expression level is correlated with the value of CAI (a CAI of 1.0 is considered to be ideal, whereas a CAI of >0.8 is rated as good for the desired expression). The ideal percentage range of GC content is between 30% and 70%. Any peaks outside this range will adversely affect transcriptional and translational efficiency.

Figure 3

Identification of boIFN–λ3/λ3^V18M. (A,B). boIFN–λ3 and boIFN–λ3^V18M SOE–PCR, respectively. M: Trans2K DNA Marker; 1~4, 5~6, 7, and 8 indicate the first, second, third, and fourth PCR results, respectively. (C). Enzyme digestion and PCR identification of the P. pastoris transformants integrated into the boIFN–λ3 gene. M. Trans2K PlusII DNA Marker; 1 and 5. PCR result based on P1/P14; 2 and 6. Xho I digested; 3 and 7 Xho I and Xba I digested; 4 and 8. PCR result based on 5′AOX/P14. 1~4 was the result of recombinant expression vector pPICZαA–boIFN–λ3 and 5~8 indicated the pPICZαA–boIFN–λ3^V18M.

Figure 4

Selection of highly expressed recombinant GS115–pPICZαA–boIFN–λ3. The numbers 1, 2, 3, 5, 6, and 13 indicate recombinant GS115–pPICZαA–boIFN–λ3, and NC indicates GS115–pPICZαA. (A). Identification results of expressed recombinant GS115–pPICZαA–boIFN–λ3 by Western blot. (B). The results of supernatants of pPICZαA–boIFN–λ3 were identified by ELISA.

Figure 5

Optimization of the expression conditions of the recombinant boIFN–λ3/λ3^V18M. (A,C) The SDS–PAGE analysis of different induction times of the recombinant boIFN–λ3 and boIFN–λ3^V18M, respectively. (B,D). The protein weight analysis of different induction times of recombinant boIFN–λ3 and boIFN–λ3^V18M, respectively. (E,F). SDS–PAGE analysis of recombinant P. pastoris GS115 under different methanol concentrations. (E). GS115–pPICZαA–boIFN–λ3. (F). GS115–pPICZαA–boIFN–λ3^V18M. M. Unstained protein molecular weight Marker; NC. The induced protein of GS115–pPICZαA.

Figure 6

Protein identification and glycoprotein analysis of recombinant boIFN–λ3/λ3^V18M. (A). SDS–PAGE for recombinant boIFN–λ3 strains 5 and 13. M. Unstained protein molecular weight Marker, 1. The induced protein of GS115–pPICZαA, 2 and 3 is the induced protein of GS115–pPICZαA–boIFN–λ3 strains 5 and 13, respectively. (B,C). Western blot analysis of rabbit anti bovine IFN–λ3 polyclonal antibody (B) and rabbit anti human IFN–λ3 polyclonal antibody (C) for immune response of recombinant boIFN–λ3. M. PageRuler Marker; 1. The induced protein of GS115–pPICZαA; 2,3. The induced protein of GS115–pPICZαA–boIFN–λ3 strains 5 and 13, respectively. (D). SDS–PAGE for recombinant boIFN–λ3^V18M strain 2. M. Unstained protein molecular weight Marker; 1. The induced protein of GS115–pPICZαA–boIFN–λ3^V18M strain 2. (E,F). Western blot analysis of rabbit anti bovine IFN–λ3 polyclonal antibody (E) and rabbit anti human IFN–λ3 polyclonal antibody (F) for immune response of recombinant boIFN–λ3^V18M. M. PageRuler Marker; 1. The induced protein of GS115–pPICZαA; 2. The induced protein of GS115–pPICZαA–boIFN–λ3^V18M strain 2. (G). SDS–PAGE analysis of the purification of recombinant boIFN–λ3 and boIFN–λ3^V18M. (H). Glycoprotein staining of recombinant boIFN–λ3 and boIFN–λ3^V18M.

Figure 7

Antiviral activity analysis of the recombinant boIFN–λ3/λ3^V18M (A) and antibody neutralization test for the recombinant boIFN–λ3 (B) and boIFN–λ3^V18M (C).

Figure 8

The characteristics of recombinant boIFN–λ3 and boIFN–λ3^V18M. (A). The results of the thermal stability test of the recombinant protein. (B). Acid and alkali resistance of the recombinant protein. (* p–value < 0.05; ** p–value < 0.01).

Figure 9

Antiproliferative analysis of boIFN–λ3/λ3^V18M.