ABSTRACT
IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P. pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.
ABSTRACT
Objective:To construct an E.coli expressing system of human interferon-?(IFN-?).Methods:Extracted DNA from human blood and PCR human IFN-?, cloned the human IFN-? gene into plasmid T-easy and pBV-220. Expressed human IFN-? in E.coli DH5?, the expressing product was analysed by SDS-PAGE, Western blot and anti-virus capacity test.Results:DNA sequence analysis showed the recombinant plasmid pBV- IFN-? contained human IFN-?. SDS-PAGE and Western blot proved that there were hIFN-? in E.coli DH5? after temperature inducing and the expressing product has anti-virus activity.Conclusion:A human IFN-? E.coli expressing system was constructed successfully, and the recombination human IFN-? has anti-virus activity.
ABSTRACT
The E. coli BL21(DE3) strain bearing the plasmid(T_7lac/His6-tag) with the h?TNF?recombinant gene was grown in LB medium containing SO?g/ml kanamycin, at 37 ℃ , up to the late logarithmic phase(OD590 ~ 0.5)then induced with IPTG (final concentration lmmol/L)for 5 hours. SDS-PAGE analysis revealed that expression level of the product(His6-?TNF?was up to 45% of the total bacterial proteins and was mainly as insoluble inclusion bodies ( IBs) . After cell lysis, the IBs was separated by centrifugation, dissolved in 7mol/L urea, then purified by Ni column ( Ni~(2+) -Sepharose 6B) . And the purity of more than 95% and the recovery of about 90% were obtained. The purified product was refolded with the renaturation buffer under low temperafure(