Shuang-Quan Zhang

Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori

The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic α-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni2+-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K12D31, Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

Lipopolysaccharide neutralization by the antibacterial peptide CM4

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. Binding of LPS to the CD14+ murine macrophage cell line RAW264.7 results in pro-inflammatory cytokine secretion. In extreme cases, it leads to septic shock in vivo. Therefore, the pursuit for molecules with antiendotoxin properties is urgent. In this study, we investigated the efficacy of antibacterial peptide CM4 in binding Escherichia coli LPS in vitro. CM4 avidly bound to E. coli LPS, as proven by the limulus amoebocyte lysate assay. Furthermore, the killing activity of CM4 against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptides affinity for endotoxin. Flow cytometric analysis revealed that CM4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, the inhibition of peptide to LPS-dependent cytokine induction was analyzed. CM4 suppressed LPS-induced TNF-alpha and IL-6 mRNA expression and blocked release of TNF-alpha and NO following LPS challenge in RAW264.7 cells. Together these observations indicate that antibacterial peptide CM4 probably exerts protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells.

Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52xa0kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.

Antifungal mechanism of antibacterial peptide, ABP-CM4, from Bombyx mori against Aspergillus niger

Antibacterial peptide, CM4 (ABP-CM4), a 35 amino acid peptide from Chinese silkworm—Bombyxxa0mori, displayed a strong antifungal activity against Aspergillusxa0niger, Trichodermaxa0viride and Gibberellaxa0saubinetii. Scanning electron microcopy showed that the morphology of conidia became more irregular and swelled when treated with ABP-CM4 at its minimal inhibitory concentration (MIC) of 8xa0μM. A cell wall regeneration assay indicated that the plasma membrane was the prime target of ABP-CM4 action. Confocal laser scanning microscopy showed that the cytoskeleton of A.xa0niger was destroyed when treated with ABP-CM4 at 8xa0μM. Furthermore, transmission electron microscopy showed that the membrane and the cellular organelles of fungus were disrupted and there were many vacuoles in the fungal cellular space after the treatment with ABP-CM4. A gel-retardation assay showed that ABP-CM4 bound the DNA of A.xa0niger. Our results suggest that ABP-CM4 exerts its antifungal activity by disrupting the structure of cell membranes and the cytoskeleton and interacts with the organelles, such as the mitochondrion and with the DNA in the fungal cell, subsequently resulting in cell death.

Molecular cloning and expression analysis of porcine γ-interferon-inducible lysosomal thiol reductase (GILT)

A porcine interferon-gamma-inducible lysosomal thiol reductase (GILT) cDNA, designated pGILT, was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) strategies. The full-length cDNA of pGILT consists of 1,062 bp with a 741 bp open reading frame, encoding 246 amino acids, with a putative molecular weight of 29.5 kDa. The deduced pGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The genomic DNA sequence of pGILT contains seven exons and six introns, which is similar to vertebrate GILT exon-intron organization. The result of real-time PCR showed that GILT is expressed in many tissues in the pig, including spleen, liver, lung, heart, intestine, blood and kidney. And the pGILT expression is obviously up-regulated in spleen and blood after induction with LPS. These results suggesting that pGILT is highly likely to play a role in the innate immune responses in porcine. It also provided the basis for investigations on the role of GILT in this important domestic species and an animal model for human diseases.

The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein

A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43xa0kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His6 and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs’-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.

Molecular structure, expression, cell and tissue distribution, immune evolution and cell proliferation of the gene encoding bovine (Bos taurus) TNFSF13 (APRIL)

A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine A Proliferation-Inducing Ligand belonging to TNF family (bAPRIL). The open reading frame (ORF) of this cDNA covers 753 bp, encoding 250 amino acids. The functional soluble part of bAPRIL (bsAPRIL) shows 97% and 92% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bAPRIL genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size, and maps to bovine chromosome 19q. Real-time quantitative PCR (qPCR) analysis revealed that bAPRIL is predominantly expressed in bovine lymphoid tissues spleen. The predicted three-dimensional (3D) structure of the bsAPRIL monomer analyzed by comparative protein modelling revealed that it is very similar to its mouse counterpart. The bsAPRIL and EGFP/bsAPRIL were efficiently expression in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, the EGFP/bsAPRIL fusion protein obtained similar fluorescence spectrum to those of EGFP. Laser scanning confocal microscopy analysis showed EGFP/bsAPRIL could bind to its receptor. In vitro, bsAPRIL could promote the proliferation of bovine or mouse splenic B cells together with/without SAC or anti-mouse IgM. Furthermore, compared to mouse soluble APRIL, the bovine soluble APRIL has the similar proliferation to mouse B cell. Those findings indicated that bsAPRIL plays an important role in proliferation of bovine B cells and has functional cross-reactivity among cow and other mammalians. Therefore, APRIL may be a potential immunologic factor for enhancing immunological efficacy in animals.

Expression and purification of antimicrobial peptide CM4 by Npro fusion technology in E. coli.

Antimicrobial peptide CM4 is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant CM4 have been reported. In order to establish a bacterium-based CM4 production system, CM4 was cloned into pET28a and expressed with Npro mutant (EDDIE) fusion. CM4 expressed as EDDIE are deposited as inclusion bodies. On in vitro refolding by switching from chemotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving CM4 protein with an authentic N terminus. Purified CM4 was separated on Ni2+-chelating chromatography column and cation-exchange chromatography column. Mass spectroscopic analysis indicated the protein to be 4132.56 Dalton, which equalled the theoretically expected mass. N-terminal sequencing of CM4 showed the sequence corresponded to the native protein. The recombinant CM4 exhibited the same antimicrobial and anti-tumor activity as reported previously. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant CM4 with native sequences.

Development of a compact anti-BAFF antibody in Escherichia coli.

Recombinant antibodies, especially single-chain antibody fragment (scFv), can be applied as detection reagents and even substitute for some reagents used in immunoassays. For scFv fragments, there is no such universal system available up to now. We have constructed vectors for the convenient, rapid expression of a novel compact antibody composed of anti-B-cell-activating factor of the TNF family (BAFF) scFv and the Fc portion (the hinge region, CH2, and CH3 domains) of the human IgG1 in Escherichiacoli. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The scFv-Fc antibody was demonstrated to retain high binding affinity to antigen, including membrane-bound BAFF and soluble BAFF, and to possess some human IgG crystallizable fragment domain functions, such as human complement C1q and protein A binding. Both size-exclusion high-performance liquid chromatography column analysis and Western blotting of proteins subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that scFv-Fc antibody is homodimeric with relative molecular mass of 110xa0kDa. These findings suggest that the compact antibody may be useful in diagnostic application for the prediction of BAFF relevant to autoimmune diseases in human.

Molecular cloning, expression, and bioactivity of dove B lymphocyte stimulator (doBAFF)

B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is a novel member of the tumor necrosis factor ligand family and plays an important role in B lymphocyte maturation and survival. cDNA of dove B lymphocyte stimulator (doBAFF) was amplified from total RNA of dove spleen by RT-PCR (reverse transcription PCR). The open reading frame of doBAFF consists 867 bases encoding a protein of 288 amino acids. Sequence comparison indicated the amino acid sequence of doBAFF showed high identity to hBAFF (50.66%) and cBAFF (91.32%). The result of RT-PCR showed that doBAFF was highly expressed in the spleen and bursa of fabricius. To enhance the soluble expression of doBAFF in Escherichia coli, we fused the extracellular region of doBAFF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fused protein SUMO-sdoBAFF was highly expressed in DE3(BL21) with a molecular weight of 35kDa. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease, Ulp1. The sdoBAFF protein was further purified by Ni-NTA affinity chromatography. In vitro, the MTT assays indicated that the purified doBAFF as well as SUMO-sdoBAFF proteins were able to promote bursa lymphocyte survival in dose-dependent manner.