tetano
Editor, Senior Moderator
Appl Biochem Biotechnol. 2016 Apr 4. [Epub ahead of print]
[h=1]Production of Influenza Virus HA1 Harboring Native-Like Epitopes by Pichia pastoris.[/h] Lin Q[SUP]1[/SUP], Yang K[SUP]1[/SUP], He F[SUP]1[/SUP], Jiang J[SUP]1[/SUP], Li T[SUP]1[/SUP], Chen Z[SUP]2[/SUP], Li R[SUP]1[/SUP], Chen Y[SUP]1,[/SUP][SUP]2[/SUP], Li S[SUP]3,[/SUP][SUP]4[/SUP], Zhao Q[SUP]5[/SUP], Xia N[SUP]1,[/SUP][SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.
[h=4]KEYWORDS:[/h] Fed-batch fermentation; Hemagglutinin; Monoclonal antibody; Native epitopes; Pichia pastoris
PMID: 27040529 [PubMed - as supplied by publisher]
[h=1]Production of Influenza Virus HA1 Harboring Native-Like Epitopes by Pichia pastoris.[/h] Lin Q[SUP]1[/SUP], Yang K[SUP]1[/SUP], He F[SUP]1[/SUP], Jiang J[SUP]1[/SUP], Li T[SUP]1[/SUP], Chen Z[SUP]2[/SUP], Li R[SUP]1[/SUP], Chen Y[SUP]1,[/SUP][SUP]2[/SUP], Li S[SUP]3,[/SUP][SUP]4[/SUP], Zhao Q[SUP]5[/SUP], Xia N[SUP]1,[/SUP][SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.
[h=4]KEYWORDS:[/h] Fed-batch fermentation; Hemagglutinin; Monoclonal antibody; Native epitopes; Pichia pastoris
PMID: 27040529 [PubMed - as supplied by publisher]