Effect of a Bombyx mori Protein Disulfide Isomerase on Production of Recombinant Antibacterial Peptides

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  • ABSTRACT

    The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.


  • KEYWORD

    Baculovirus Expression Vector System (BEVS) , Bombyx mori , Antibacterial peptide , Protein Disulfide Isomerase (PDI)

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  • [Fig. 1.] Construction of baculovirus transfer vector for production of chemeric mature nuecin and enbocin fused bPDI lacking the ER retention signal KDEL. The open reading frame of bPDI lacking the ER retention signal KDEL (bPDI-KDEL) was subcloned into the BamHI and StuI site in baculovirus transfer vector pBAC-1. The coding sequence of mature nuecin (mNuecin) and enbocin (mEnbocin) linked factor Xa cleavage site was flanked by StuI and XhoI site, and then inserted into the pBAC1-(bPDI-KDEL) vector with StuI and XhoI. The baculovirus transfer vector pBAC1-(bPDI-KDEL)-mNuecin-mEnbocin was disgested with BamHI/XhoI (lane 1) and BamHI/ StuI/ HindIII/XhoI (lane 2). M, 1 kb ladder DNA markers.
    Construction of baculovirus transfer vector for production of chemeric mature nuecin and enbocin fused bPDI lacking the ER retention signal KDEL. The open reading frame of bPDI lacking the ER retention signal KDEL (bPDI-KDEL) was subcloned into the BamHI and StuI site in baculovirus transfer vector pBAC-1. The coding sequence of mature nuecin (mNuecin) and enbocin (mEnbocin) linked factor Xa cleavage site was flanked by StuI and XhoI site, and then inserted into the pBAC1-(bPDI-KDEL) vector with StuI and XhoI. The baculovirus transfer vector pBAC1-(bPDI-KDEL)-mNuecin-mEnbocin was disgested with BamHI/XhoI (lane 1) and BamHI/ StuI/ HindIII/XhoI (lane 2). M, 1 kb ladder DNA markers.
  • [Fig. 2.] SDS-PAGE of cell lysates (A) and Western blots of cell culture media (B) for chimeric nuecin and enbocin fused with bPDI-KDEL. Sf9 cells (3.0×106) were infected with recombinant baculovirus [vAc-(bPDI-KDEL)-mNuecin-mEnbocin; lane 3)] encoding (bPDI-KDEL)-mNuecin-mEnbocin-His6. Cells and cell culture media were harvested 96 h after infection (A). Western blots were performed using His6-tag antibody (B). Lane 1, proteins extracted from normal cells; lane 2, proteins extracted from cells infected with wild-type baculovirus. Arrows indicate the putative (bPDI-KDEL)-mNuecin-mEnbocin band.
    SDS-PAGE of cell lysates (A) and Western blots of cell culture media (B) for chimeric nuecin and enbocin fused with bPDI-KDEL. Sf9 cells (3.0×106) were infected with recombinant baculovirus [vAc-(bPDI-KDEL)-mNuecin-mEnbocin; lane 3)] encoding (bPDI-KDEL)-mNuecin-mEnbocin-His6. Cells and cell culture media were harvested 96 h after infection (A). Western blots were performed using His6-tag antibody (B). Lane 1, proteins extracted from normal cells; lane 2, proteins extracted from cells infected with wild-type baculovirus. Arrows indicate the putative (bPDI-KDEL)-mNuecin-mEnbocin band.
  • [Fig. 3.] Antibacterial activity of recombinant nuecin and enbocin protein against Escherichia coli. 5 ml of culture medium (2 × 106 cells ml-1) were concentrated to 500 ml using a freezing dryer; 40 ml of concentrated sample were loaded onto a paper disk. N, recombinant nuecin; E, recombinant enbocin; P+N+E, chemric recombinant nuecin and enbocin with bPDI-KDEL.
    Antibacterial activity of recombinant nuecin and enbocin protein against Escherichia coli. 5 ml of culture medium (2 × 106 cells ml-1) were concentrated to 500 ml using a freezing dryer; 40 ml of concentrated sample were loaded onto a paper disk. N, recombinant nuecin; E, recombinant enbocin; P+N+E, chemric recombinant nuecin and enbocin with bPDI-KDEL.
  • [Fig. 4.] Antibacterial activity of recombinant nuecin and enbocin against various plant pathogens. Five ml of culture medium (2×106 cells ml-1) were concentrated to 500 ml using a freeze-dryer, and 40 ml of concentrated samples were loaded onto paper disk. Values are the mean diameter of the clear zones ± standard deviation from three independent experiments. P < 0.05 versus control. D, distilled water (control); 1, P. syringae ; 2, P. tollasii ; 3, S. aureus; 4, A. tumefaciens ; 5, R. solacerum ; 6, E. mallotivora ; 7, P. carotovorum ; 8, E. chrysantuemi ; and 9, B. megaterium.
    Antibacterial activity of recombinant nuecin and enbocin against various plant pathogens. Five ml of culture medium (2×106 cells ml-1) were concentrated to 500 ml using a freeze-dryer, and 40 ml of concentrated samples were loaded onto paper disk. Values are the mean diameter of the clear zones ± standard deviation from three independent experiments. P < 0.05 versus control. D, distilled water (control); 1, P. syringae ; 2, P. tollasii ; 3, S. aureus; 4, A. tumefaciens ; 5, R. solacerum ; 6, E. mallotivora ; 7, P. carotovorum ; 8, E. chrysantuemi ; and 9, B. megaterium.