Expression of Huiyang chicken IFNγ gene in Pichia pastoris and analysis of antiviral activity of the recombinant protein
0
Citation
0
Reference
20
Related Paper
Abstract:
To identify antiviral activity and the secondary structure of the recombinant protein of Huiyang chicken IFN-γ (chIFN-γ2) expressed in Pichia pastoris,the cDNA encoding mature peptide of IFN-γ was subcloned from plasmid pGEM-T-chIFN-γ2 and recombined with vector pPICZαB.The recombinant plasmid confirmed by enzyme digestion,PCR amplification and sequencing was transformed into P.pastoris GS115.After methanol induction,SDS-PAGE analysis of the culture supernatant of recombinant yeast strains indicated that the yield of recombinant IFN-γ was 0.604mg/mL and the molecular weight was(approximately) 33ku.The recombinant IFN-γ expressed in P.pastoris had antiviral activity of 3.2×10~4(U/mg) in CEF-VSV.The secondary structure of the recombinant IFN-γ was investigated by Circular Dichroism,and contents of α helix,β sheet,turn,and random were 55.1%,8.3%,13.9% and 22.8%,respectively.These results showed that the recombinant IFN-γ with high antiviral activity was expressed successfully in P.pastoris and the chicken IFN-γ was a helical protein which was similar to IFN-γ of mammals.Keywords:
Pichia
Cite
In order to construct recombinant yeast strains expressing extracellular ChIFNα,the recombinant expression vector pPIC-ChIFNα of chicken interferon alpha mature protein gene was transformed into Pichia pastoris GS115 strain by electroporation to integrate with the genome,many positive Pichia pastoris strains were obtained by PCR analysis.The transformants with multicopy were screened by high concentration G418 and induced to express ChIFNα with methanol.Two integrants with high expression level were obtained,named GS-ChIFNαB_1 and GS-ChIFNαB_2.The culture supernatant of high expression strains was concentrated by PEG20000,purified with DEAE Sepharose Fast Flow and Sephadex G200,and the antiviral activity of secretion supernatant was detected by vesicular stomatitis cytopathic effect inhibition assay(CPE).The assay results revealed that the recombinant ChIFNα gene was expressed highly and secreted effectively in Pichia pastoris,and the expressed product had normal bioactivity.The antiviral activity units was 570×10~4 U/ml,and the specific activity of recombinant ChIFNα was up to 2 980×10~4 U/mg protein.
Pichia
Cite
Citations (0)
IFN-α is a cytokine with high antiviral activity. Huiyang IFN-α was cloned from liver genomic DNA. The homoligies were from 96.9%～97.9% between Huiyang chicken IFN-α and IFN-α s on Genbank, indicating this IFN-α gene was a new subtype. The recombinant plasmid IFN-α/pPICZαA confirmed by enzyme digestion, PCR and sequencing was transformed into Pichia pastoris GS115. After 1% methanol induction, SDS-PAGE analysis of the culture supernatant of recombinant yeast strains indicated that the yield of recombinant IFN-α was 0.307 mg/ml and the molecular weight was about 35 kD. The recombinant IFN-α expressed by Pichia pastoris showed antiviral activity of 3.2×106 U/mg in CEF/VSV, which was higher than that from E.Coli. The secondary structure of recombinant IFN-γ analyzed by Circular Dichroism showed that the contents of this protein were: 53.2% α-helix;3.1% β sheet, 10.6% turn; 33.1% random, which showed it was a tipical helical protein. This paper reported that recombinant IFN-α with high antiviral activity was highly produced in Pichia pastoris and first confirmed that chicken IFN-α was a helical protein which was similar to IFN-α of mammals.
Pichia
Cloning (programming)
Cite
Citations (0)
To obtain efficient expression of Bovine lysozyme-1(LYZ1),LYZ1 gene was designed and synthesized using the preference codons of Pichia pastoris,and inserted into the Pichia pastoris expression vector pPICZα-A.The recombinant plasmid pPICZ-LYZ1 was linearized and transformed into Pichia pastoris strain GS115 by electroporation.Recombinant Pichia pastoris was screened on YPDS plates containing Zeocin and identified by PCR.Expression of LYZ1 was detected by SDS-PAGE and confirmed by western blot analysis.Lysozyme activity was detected by Micrococcus lysodeikticus(ML).Result showed that recombinant protein was expressed in soluble form with a molecular weight approximately 16 ku.The biological activity were shown up to 2 842 u/mL was detected in the supernatant by ML assay.The recombinant LYZ1 had better antibacterial activity on staphylococci and E.coli,but was inferior against Streptococcus mastitidis,Streptococcus dysgalactiae and Streptococcus uberis.
Streptococcus uberis
Streptococcus dysgalactiae
Pichia
Cite
Citations (0)
AIM: To achieve high level expression and purification of recombinant mature human IL-18(mhIL-18)protein in pichia pastoris.METHODS: An Intein Tag was added to the vector.The gene of mhIL-18-Intein was amplified by SOEing and asymmetric PCR.The recombinant expression plasmid pPIC9-IL-18-Intein was constructed by cloning mhIL-18-Intein into pPIC9 vector and was transformed to GS115.The expression of recombinant fusion protein was induced by methanol under an optimum inducing condition.After the protein excreted out of the cells was harvested,SDS-PAGE and Western Blot were used to analyze the expression of recombinant fusion protein.The activity of mhIL-18 purified by affinity chromatography was measured by MTT assays.RESULTS: The cloned sequence of mhIL-18 was identical with the sequence in GenBank.After methanol induction,the fusion protein of mhIL-18 reached the secretion peak of 100 mg/L at 96 h.The purity of the recombinant expressed mhIL-18 was 95% by means of affinity chromatography and the purified mhIL-18 had the biological activity of IL-18.CONCLUSION: We have succeeded in expressing and purifying the recombinant human IL-18 with obvious biological activity in pichia pastoris.
Intein
Pichia
Myc-tag
FLAG-tag
Cloning (programming)
Cite
Citations (0)
AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His^+ Mut^+) by electroporation. His^+Mut^+ recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His^+Mut^+ after transformation of P.pastoris with linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 ml_/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in Ecoliwhen the same rSCoVN protein quantity was used.CONCLUSION: Active re
Antigenicity
Pichia
Cite
Citations (1)
An artificial gene for consensus interferon mutant was synthesized by using favored codons of the yeast Pichia pastoris . The gene was cloned into the secretory expression vector pMEX9K, and the recombinant vector pMEX CIFNm was linearized and transformed into GS115 for expression. The culture supernatant of transformants had the antiviral activity after inducement with methanol. The purity of recombinant protein reached over 95% through the steps of ion exchange, hydrophobic interaction, size exclusion chromatography. The first 5 amino acid sequence of the N terminal was consistent with the theoretical sequence. Its molecular weight measured by MS agree with the theoretical value 19 3 kD. The specific activity of protein was similar to consensus interferon(infergen)——6×10 8 antiviral units/mg when assayed by the antiviral activity of WISH cells challenged with VSV virus and Lowry protein assay.
Pichia
Antiviral protein
Cite
Citations (2)
Objective:To express and purify human interferon ω(IFN_ω) in yeast Pichia pastoris to evaluate its functions.Methods:Human IFN_ω gene, obtained by PCR_amplification, was cloned into the P.pastoris expression vector pMEX9K which has been cleaved by Xho Ⅰand Eco RⅠ.The recombinant vector pMEX9Kω was verified to be correctly constructed by enzyme digestion, PCR and sequencing.After linearization by Sal Ⅰ, the pMEX9Kω was transformed into GS115 by elctroporation. After being induced by methanol, the target protein IFN_ω was expressed in fermentation supernatant at high level. The purified protein was finally obtained with purity being higher than 99 per cent as determined by reverse phase HPLC by three_step chromatography: Butyl Sepharose 4 FF, SP Sepharose FF and Superdex 75.Results:The resulting protein was of at least 99% purity as determined by reverse phase HPLC. Analysis of the recombinant protein revealed that the protein had the same N_terminal sequence as the native one, its molecular weight was 22?000, and its specific activity was similar to natural human IFN_ω. Conclusion:The recombinant human IFN_ω was successfully expressed and purified in P.pastoris . [
Pichia
Sepharose
Cite
Citations (0)
Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR,and subcloned into Pichia pastoris expression vector pPICZαA,thus constructing the recombinant plasmid pPICZαA-ChIL-18.After identified by restriction enzymes digestion,PCR and DNA sequencing,the recombinant plasmid was transformed into P.pastoris X-33.Then the multi-copy recombinant strains were induced for expression.The expression conditions were further optimized by analyzing the influences of pH value,inducer concentration,inducing time,and inducing temperature on the yields of target protein.The results showed that chicken IL-18 could be secreted by the recombinant P.pastoris and the expression level of recombinant protein was highest under pH 6.0,1.5% of methanol,temperature 26 ℃,and 96 hours of inducing time.This study laid a foundation for the large-scale fermentation of chicken IL-18.
Pichia
Cite
Citations (0)
Full length cDNA of human IL-11 was synthesized by DNA synthesizer. An expression plasmid, pGENYk, containing the recombinant DNA fragment, was linearized and transformed into Pichia pastoris. This recombinant gene was highly expressed in this yeast, and the expression product was purified by a three-step chromatography method. Analysis of the purified recombination protein with SDS-PAGE, Western blot and biological activities showed that the activity of the protein was the same as the Neumega expressed in E.coli.
Pichia
Myc-tag
Cite
Citations (4)
Objective To obtain recombinant human interleukin 10(rhIL-10) secretorily expressed in Pichia pastoris with bioactivity for studying its biological function and animal test. Methods IL-10 gene was inserted into the sites between XhoⅠ and EcoRⅠ downstream of α factor in the recombinant plasmid α/pUC18. Then, the fusion gene αIL-10 was digested and inserted into the sites between BamHⅠ and EcoRⅠ of expression plasmid pPIC9K of P. pastoris. After the recombinant plasmid IL-10/pPIC9K was analyzed to be correct construct with digestion and sequencing, it was linearized with digestion of SalⅠ and transformed into P. pastoris strain GS115. Recombinant yeast colonies grown in auxotrophic medium MD were in vivo screened with G418 for multicopy expression cassettes. Yeast transformants were proved to have the desired gene IL-10 with PCR, and then induced for expression of recombinant human IL-10 with methanol. Recombinant human IL-10 was induced to secretorily express with methanol. The desired protein was identified with SDS-PAGE and Western blot. IL-10 was quantitatively assayed with ELISA and its biological activity was analyzed with stimulating proliferation of MC/9 cells. Results Recombinant expression plasmid IL-10/pPIC9K was successfully constructed. Four transformants were screened with G418 and PCR. Induced with methanol, the expression level of IL-10 was (0.627±0.09)mg/L at flask fermentation, and its specific activity was 1.5×10~5 U/mg. Conclusion Recombinant human IL-10 has good biological activity secretorily expressed in P. pastoris.
Pichia
Cite
Citations (1)