tetano
Editor, Senior Moderator
Biotechnol Prog. 2016 Nov 1. doi: 10.1002/btpr.2400. [Epub ahead of print]
[h=1]Insertion of single-chain variable fragment (scFv) peptide linker improves surface display of influenza hemagglutinin (HA1) on non-recombinant Lactococcus lactis.[/h] Jee PF[SUP]1[/SUP], Chen FS[SUP]1[/SUP], Shu MH[SUP]1[/SUP], Wong WF[SUP]1,[/SUP][SUP]2[/SUP], Abdul Rahim R[SUP]3[/SUP], AbuBakar S[SUP]1,[/SUP][SUP]2[/SUP], Chang LY[SUP]4,[/SUP][SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7?1.4% and 3594.0?675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1?1.5% and 1652.0?34.1, respectively). Higher amount of HA1/L/AcmA (∼2.9 ? 10[SUP]4[/SUP] molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (∼1.1 ? 10[SUP]4[/SUP] molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. This article is protected by copyright. All rights reserved.
? 2016 American Institute of Chemical Engineers.
[h=4]KEYWORDS:[/h] N-acetylmuramidase; biotechnology; influenza virus; scFv peptide linker; surface display
PMID: 27802566 DOI: 10.1002/btpr.2400
[PubMed - as supplied by publisher]
[h=1]Insertion of single-chain variable fragment (scFv) peptide linker improves surface display of influenza hemagglutinin (HA1) on non-recombinant Lactococcus lactis.[/h] Jee PF[SUP]1[/SUP], Chen FS[SUP]1[/SUP], Shu MH[SUP]1[/SUP], Wong WF[SUP]1,[/SUP][SUP]2[/SUP], Abdul Rahim R[SUP]3[/SUP], AbuBakar S[SUP]1,[/SUP][SUP]2[/SUP], Chang LY[SUP]4,[/SUP][SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51.7?1.4% and 3594.0?675.9, respectively) in comparison to the HA1/AcmA without the scFv peptide linker (41.1?1.5% and 1652.0?34.1, respectively). Higher amount of HA1/L/AcmA (∼2.9 ? 10[SUP]4[/SUP] molecules per cell) was displayed on L. lactis when compared to HA1/AcmA (∼1.1 ? 10[SUP]4[/SUP] molecules per cell) in the immunoblotting analysis. The HA1/L/AcmA completely agglutinated RBCs at comparable amount of protein to that of HA1/AcmA and HA1. Computational modeling of protein structures suggested that scFv peptide linker in HA1/L/AcmA kept the HA1 and the AcmA domain separated at a much longer distance in comparison to HA1/AcmA. These findings suggest that insertion of the scFv peptide linker between HA1 and AcmA improved binding of recombinant proteins to L. lactis. Hence, insertion of scFv peptide linker can be further investigated as a potential approach for improvement of heterologous proteins displayed on the surface of L. lactis using the AcmA binding domain. This article is protected by copyright. All rights reserved.
? 2016 American Institute of Chemical Engineers.
[h=4]KEYWORDS:[/h] N-acetylmuramidase; biotechnology; influenza virus; scFv peptide linker; surface display
PMID: 27802566 DOI: 10.1002/btpr.2400
[PubMed - as supplied by publisher]