Showing papers on "XhoI published in 2008"

An improved SUMO fusion protein system for effective production of native proteins

Abstract: Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.

Whole-Genome Amplification by Degenerate Oligonucleotide Primed PCR (DOP-PCR).

Abstract: INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. Unlike other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). The degenerate-oligonucleotide-primed PCR (DOP-PCR) method described here allows complete genome coverage in a single reaction. In contrast to the pairs of target-specific primer sequences used in traditional PCR, only a single primer, which has defined sequences at its 5'-end (containing an XhoI restriction site) and 3'-end and a random hexamer sequence between them, is used here. DOP-PCR comprises two different cycling stages. In stage 1 (low stringency), low-temperature annealing and extension in the first five to eight cycles occurs at many binding sites in the genome. The 3'-end of the primer binds at sites in the genome complementary to the 6-bp well-defined sequence at the 3'-end of the primer (~10(6) sites in the human genome). The adjacent random hexamer sequence (displaying all possible combinations of the nucleotides A, G, C, and T) can then anneal and tags these sequences with the DOP primer. In stage 2 (high stringency; >25 cycles), the PCR annealing temperature is raised, which increases priming specificity during amplification of the tagged sequence. DOP-PCR generates a smear of DNA fragments (200-1000 bp) that are visible on an agarose gel.

RFLP analysis of Hordeum species relationships1

Abstract: Detailed ribosomal DNA restriction maps were prepared for eight representative plants from Hordeum L. Section Hordeum (two H. bulbosum, H. glaucum, a diploid H. leporinum, a tetraploid H. leporinum, H. murinum, H. spontaneum, H. vulgare) in order to explore species relationships. The four restriction enzymes (EcoRI,SacI, BamHI and HindIII) used in our earlier study were supplemented with six additional ones (BglI, ClaI, KpnI, Pvu II, Sst II and XhoI). Southern blots prepared, using the restriction enzymes singly and in combinations, were probed with the wheat ribosomal probe pTA7l, and hybridization patterns analyzed. A comparison of the derived maps with known maps for wheat, pea and pumpkin showed strong conservation of restriction sites, especially in the ribosomal coding region. The maps suggest that duplication of coding region DNA and its insertion into the spacer region as well as amplification of subrepeats are responsible for variation in rDNA repeat length. Phylogenetic analysis indicated the early divergence of ancestral wheat and Hordeum species, followed by the divergence of the progenitors of H. spontaneum/H. vulgare and the remaining Hordeum species and, most recently, between those of H. glaucum and H. murinum.

DNA molecule manipulation by motor proteins for analysis at the single-molecule level

Abstract: Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale.

Immunogenicity and immunoprotection of recombinant PEB1 in Campylobacter-jejuni-infected mice.

Abstract: AIM: To construct a prokaryotic expression vector carrying Campylobacter jejuni peb1A gene and express it in Escherichia coli. Immunoreactivity and antigenicity of rPEB1 were evaluated. The ability of rPEB1 to induce antibody responses and protective efficacy was identified. METHODS: peb1A gene was amplified by PCR, target gene and prokaryotic expression plasmid pET28a (+) was digested with BamHI and XhoI, respectively. DNA was ligated with T4 DNA ligase to construct recombinant plasmid pET28a(+)-peb1A. The rPEB1 was expressed in E. coli BL21 (DE3) and identified by SDS-PAGE. BALB/c mice were immunized with rPEB1. ELISA was used to detect the specific antibody titer and MTT method was used to measure the stimulation index of spleen lymphocyte transformation. RESULTS: The recombinant plasmid pET28a (+)-peb1A was correctly constructed. The expression output of PEB1 protein in pET28a (+)-peb1A system was approximately 33% of total proteins in E. coli. The specific IgG antibody was detected in serum of BALB/c mice immunized with rPEB1 protein. Effective immunological protection with a lower sickness incidence and mortality was seen in the mice suffering from massive C. jejuni infection. CONCLUSION: rPEB1 protein is a valuable candidate for C. jejuni subunit vaccine.

Restriction on conjugational transfer of pLS20 in Bacillus subtilis 168

Abstract: Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.

Cloning and prokaryotic expression of C‐type lysozyme gene from agrius convolvuli

Abstract: We have isolated and characterized Agrius convolvuli cDNA encoding a c‐type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino‐terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21 (DE3) pLysS cells for pGEX 4T‐1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T‐1 vector, which contained the glutathione Stransferase (GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS‐PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was...

Expression of bovine leukemia virus p24 protein in bacterial cell.

Abstract: Bovine leukemia virus (BLV) is a member of the family Retroviridae, genus Deltaretrovirus that has three important gene including gag, pol and env. This virus causes B-cell lymphocytosis and lymphosarcoma in cows. In the first step PCR product of gag gene of BLV isolated in different regions of Iran and BLV-FLK strain were cloned in to a pTZ57R/T vector, then insert were digested by BglII and XhoI restriction enzymes and cloned in to pET-28(a) as an expression vector. For the expression of p24 protein, the pET-28(a) recombinant vector was transformed in BL21(DE3) strain of E. coli competent cell and after induction of the protein having been expressed by IPTG, the presence of gag expressed protein was shown in immunoblotting and SDS-PAGE system. With respect to the remarkable frequency of infection to BLV in Iran and the necessity of controlling it through vaccination with recombinant vaccines of gp51, manufacturing and applying the recombinant p24 protein are vital goals in recognition and distinction between infection and responses caused by vaccine.

[Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

Abstract: This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.