Construction Of PT16-SPM Fusion Expression Vector And Expression And Purification Of Bovine Recombinant IFN-?

Purification of recombinant proteins is a major challenge in protein function study and drug development. Presently, affinity chromatography is the most popular method for recombinant protein purification with high purification efficiencies, but these chromatographic methods require expensive resins and are difficult to scale-up. In addition, the affinity tags have generally to be removed from the final products. This can be achieved by digestion with endopeptidases, which are expensive, removed from the final product and thus complicate the purification process further. Therefore, simple and economical purification methods are urgently needed for recombinant protein preparation, for animal use in particular.The self-processing module (SPM) of Neisseria meningitides FrpC protein consists of 250 amino acids, which can undergo self-cleavage at Asp414/Pro415 under induction of calcium. By combining the self-cleaving activity of SPM with the temperature-sensitive reverse transition of elastin-like polypeptides (ELP), a simple economical expression and purification system has been established in our research group. Self-assembling peptides can form inclusion body-like aggregates in bacterial cells, but their fusion expression products are active proteins which can be purified with simple methods such as centrifugation. By combining the self-cleaving activity of SPM with a self-aggregation tag, in this study we established an even more simple recombinant protein expression and purification system, and recombinant bovine interferon-y (BoIFN-y) was successfully expressed and purified with the system established.The coding sequence for self-aggregation peptide ELK16 was adapted to E. coli codon usage using JAVA Codon Adaption Tool and fused C-terminally with a PT linker consisting of 17 proline (P) or threonine (T) residues. The synthetic fusion sequence PT16 was cloned into prokaryotic expression vector pET-30a. The coding sequence was amplified using high fidelity PCR and fused V-terminally to the PT16 sequence, resulting expression vector pSPM-PT16. The coding sequence for the mature peptide of BoIFN-y was subcloned into the pSPM-PT16 vector as an NdeI/SalI segment and the recombinant vector was transformed to E. coli strain BL21 (DE3) for expression. By systematic optimization of the expression conditions such as medium, IPTG concentration, induction temperature and time, the BoIFN?-SPM-PT16 fusion protein was efficiently expressed. After optimization of the purification conditions such as centrifugation force, Triton X100 wash concentration and denaturation/renaturation with urea, the fusion protein was purified to a purity of 92.7% with a recovery rate of 70.4%. After calcium-activated self-cleavage of fusion protein, the recombinant BoIFN? was efficiently recovered by an additional round of centrifugation with a purity of 94.9%. By using MDBK/VSV system, cytopathic effect protection assay showed that recombinant BoIFN-? has a specific bioactivity of 103 U/mg.