Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis

Abstract

Anti-lipopolysaccharide factors (ALFs) with a LPS-binding domain (LBD) are considered to have broad spectrum antimicrobial activities and certain antiviral properties in crustaceans. FcALF2 was one isoform of ALFs isolated from the Chinese shrimp Fenneropenaeus chinensis. Our previous study showed that a modified LBD domain (named LBDv) of FcALF2 exhibited a highly enhanced antimicrobial activity. In the present study, a modified FcALF2 gene (mFcALF2), in which the LBD was substituted by LBDv, was designed and synthesized. This gene was successfully expressed in yeast Pichia pastoris GS115 eukaryotic expression system, and the characteristics of the recombinant protein mFcALF2 were analyzed. mFcALF2 exhibited apparent antibacterial activities against Gram-negative bacteria, including Escherichia coli, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, and Gram-positive bacteria, including Bacillus licheniformis and Staphylococcus epidermidis. In addition, mFcALF2 could reduce the propagation of white spot syndrome virus (WSSV) in vivo by pre-incubation with virus. The present study paves the way for developing antimicrobial drugs in aquaculture.

Keywords: anti-lipopolysaccharide factors; antibacterial activity; antiviral activity; recombinant protein.

Figure 1

The nucleotide sequence and its deduced amino acid sequence of the modified anti-lipopolysaccharide factor isoform 2 from Fenneropenaeus chinensis (FcALF2) gene (mFcALF2). (A) Schematic representation of the vector pPIC9K-mFcALF2; (B) The LBD region of mFcALF2 is shown in bold and the stop codon is indicated by an asterisk. The restriction enzyme sites are underlined. The 6× His-tag is shown in box; (C) Multiple sequence alignment among mFcALF2, FcALF2 and LBDv.

Figure 2

Detection of the recombinant mFcALF2 protein. (A) SDS-PAGE analyses of the recombinant mFcALF2 protein. Lane M in A and B represent molecular mass standards. Lane 1 shows the concentrated protein in supernatant secreted in GS115. Lane 2 shows the purified mFcALF2 protein; (B) Western blot analysis of the recombinant protein by anti-His tag antibody.

Figure 3

Molecular weight analysis of the recombinant mFcALF2 protein by MALDI/TOF (matrix-assisted laser desorption ionization mode) mass spectrometry.

Figure 4

Binding activity analysis of recombinant mFcALF2 to bacteria. Star (*) indicates significant differences (p < 0.05) between the treated and untreated groups of different bacteria. The data are analyzed based on ANOVA with post hoc.

Figure 5

Morphology of bacteria after treatments by recombinant mFcALF2. The 10⁸ cfu/mL different bacteria are incubated with 32 μM LBDv peptide for 2 h. The bacteria treated with same concentration pGFP peptide are used as negative control. Bar scale is 1 μM.

Figure 6

Inhibition zones of recombinant mFcALF2 to different bacteria: (A) E. coli; (B) V. anguillarum; (C) B. licheniformis; and (D) S. epidermidis. ‘‘Blank’’ represents blank group with nothing added. ‘‘PBS’’ represents control group with only PBS. ‘‘pGFP’’ represents negative control with synthetic pGFP peptide. “LBDv” represents positive control with synthetic LBDv peptide. mFcALF2 represents the recombinant protein. Twenty microliters of 32 μM protein/peptide solution is added to the center of filter paper.

Figure 7

Hemolytic phenotypes of mFcALF2 on sheep blood agar. The same amount of PBS buffer (pH 7.4) and 0.2% Triton X-100 were used as negative and positive controls, respectively. The 60 μL purified mFcALF2 protein (32 μM) was added into the Oxford cup.

Figure 8

Detection of viral loads in L. vannamei after injection of WSSV incubated with recombinant mFcALF2. Data represent the means ± S.E. Lowercase letters (a, b, c, d and e) represent significant difference among treatments at p < 0.05. Three replicate experiments are performed. The data were analyzed based on ANOVA with post hoc.