FGD2, a member of FGD family, contains a Dbl homology domain (DH) and two pleckstrin homology domains segregated by a FYVE domain. The DH domain has been deduced to be responsible for guanine nucleotide exchange in CDC42 to activate downstream factors. Our aim was to build a prokaryotic expression system for the DH domain and to examine its guanine nucleotide exchange activity toward CDC42 in vitro.
A recombinant vector, which was successfully constructed based on pGEX-6P-1, was employed to express the DH domain of human FGD2 (FGD2-DH) in E.coli BL21 (DE3). Purified FGD2-DH behaved as a homogeneous monomer with an estimated molecular weight that corresponded to the theoretical molecular weight and was predicted to be an α-helix protein by circular dichroism spectroscopy. joplink Recombinant Human Aquaporin-1 FGD2-DH displayed weak guanine nucleotide exchange activity in vitro and very weak interactions with CDC42 following glutaraldehyde cross-linking.
Delivery of HIV-1 polyepitope constructs using cationic and amphipathic cell penetrating peptides into mammalian cells
Improving an effective vaccine against human immunodeficiency virus 1 (HIV-1) is an important global health priority. Despite many efforts in development of the HIV-1 vaccine, no effective vaccine has been approved yet. Recently, polyepitope vaccines including several immunogenic and conserved epitopes of HIV-1 proteins have received a special attention.
In this study, HIV-1 Nef, Tat, Gp160 and P24 proteins were considered for the selection of immunodominant and conserved epitopes due to their critical roles in the viral life cycle and pathogenesis. At first, the Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA construct was designed using in silico studies.
Then, the DNA construct was subcloned in pEGFP-N1 and pET-24a (+) expression vectors and the rNef-Tat-Gp160-P24 polyepitope peptide was generated in E.coliexpressionsystem for in vitro delivery using novel cell-penetrating peptides (CPPs), LDP-NLS and CyLoP-1, in a non-covalent manner. Also, the HR9 and MPG CPPs were used to transfer the DNA construct.
Our results showed that the recombinant polyepitope peptide generated in Rosetta strain migrated as a clear band of ~31 kDa in SDS-PAGE. The SEM data confirmed the formation of stable nanoparticles with a size below 250 nm. MTT assay revealed that the complexes did not represent any considerable cytotoxic effect compared to untreated cells.
The results of fluorescence microscopy, flow cytometry, and western blotting indicated that these CPPs successfully delivered polyepitope constructs into HEK-293T cell line. These data suggested that these CPPs can be used as a promising approach for the development of the HIV-1 vaccine.
Molecular characterization and complement activating functional analysis of a new collectin(TfCol-11) from Trachidermus fasciatus.
Molecular characterization and complement activating functional analysis of a new collectin(TfCol-11) from Trachidermus fasciatus
The complement system is a crucial component of the innate immune system that links innate and adaptive immunity. CL-11, a protein similar to Mannose-binding lectin (MBL), plays significant role in the innate immune system in mammals and fish, serving as an initiator of the lectin pathway of complement activation. In this study, a CL-11 homolog (TfCol-11) was identified in roughskin sculpin (Trachidermus fasciatus), and its expression and role in immune responses were characterized. The open reading frame of TfCol-11 is 795 bp long, encoding a 264 amino acid polypeptide.
The deduced amino acid sequence of this protein is highly homologous to sequences in other teleosts, and is similar to vertebrate CL-11, containing a canonical collagen-like region, a carbohydrate recognition domain, and a neck region. Recombinant TfCol-11 purified from Escherichia coli(E.coli) was able to bind to different microbes in a Ca2+-independent manner. Meanwhile, a 993 bp-long of partial MASP cDNA with a 96 bp 5′ untranslated region (UTR) was also cloned from roughskin sculpin, containing 299 amino acids and consisting of three domains (CUB-EGF-CUB). qRT-PCR indicated that TfCol-11 and MASP mRNAs were predominately co-expressed in the liver.
The temporal expression of TfCol-11 and MASP were both drastically up-regulated in the liver, skin, and blood by LPS challenge. Recombinant TfCol-11 purified from E.coli BL21(DE3) was able to agglutinate some bacteria in a Ca2+-dependent manner. In addition, an in vitro pull-down experiment demonstrated that TfCol-11 was able to bind to MASP, and in vivo experiments showed that TfCol-11 was associated with increased membrane attack complex (MAC) levels. It is therefore possible that TfCol-11 may plays a role in activating the complement system and in the defense against invading microorganisms in roughskin sculpin.
Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain
The early developmental stages of the mud crab Scylla paramamosain suffer from high mortality caused by pathogen infections; however, few immune associated factors are known. Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) functions as a typical pathogen recognition receptor and plays an important role in the innate immune system of invertebrates. In this study we characterized a LGBP gene (SpLGBP) which was highly expressed in the late embryonic, zoea I larval stage and hepatopancreas of S. paramamosain.. It encodes 364 amino acids, composed of several conserved domains like the bacterial glucanase motif.
The recombinant SpLGBP protein (rSpLGBP) was obtained through the E.coli expression system, in which two 6◊His-tags were added to both C and N terminals during vector construction for the improvement of purification efficiency. In vivo the study showed that the SpLGBP mRNA was significantly up-regulated under Vibrio parahaemolyticus and a lipopolysaccharide (LPS) challenge in the hemocytes and hepatopancreas.
The ELISA binding assay in vitro indicated that the rSpLGBP was capable of binding to LPSs and peptidoglycan (PGN). The rSpLGBP could agglutinate both G+ and G- bacteria in the presence of Ca2+. Our results suggest that SpLGBP may play an immunological role against pathogenic infection in the early developmental stages of S. paramamosain.
Characterization of the LECT2 gene and its protective effects against microbial infection via large lymphocytes in Lampetra japonica
Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional protein of the innate immune system that defends against bacterial infections and chemotactic activity. However, its precise function in lamprey remains unclear. In this study, a novel LECT2 gene was first cloned from Lampetra japonica. The full-length cDNA sequence of L-LECT2 consists of a 606-bp ORF encoding a protein of 201 amino acid residues. L-LECT2 has greater than 50% sequence identity with its homologs in jawed vertebrates.
FACS and immunohistochemistry assays were used to determine that the L-LECT2 protein was primarily distributed in the intestines and supraneural body tissues of lamprey, also marginally detectable in leukocytes. However, the expression of L-LECT2 was differentially upregulated in the intestines and heart after treatment with LPS.
The recombinant L-LECT2 resulted in significant promoting migration of the leukocytes in vitro. Our data demonstrate that L-LECT2 treatment could enhance phagocytosis in lamprey large lymphocytes. Thus, our results suggest that LECT2 can modulate the host defense in lamprey and mediate antibacterial protection against E.coli through large lymphocytes.