Prokaryotic Expression

blot, indicating that the fusion protein had high immu- noreactivity. However, we could not explain presence of lower intensity bands other than that of the right sized band, both in SDS-PAGE (Figure 3B) and their subse- quent recognition in Western Blot (Figure 4). Perhaps these are E. coli BL21 (DE3) proteins which cross- reacted with the commercial rabbit antiserum against the whole cell of H. pylori. Several other approaches have been used for construction and use of prokaryotic expression system for potential H. pylori vaccine candidates like flaA, flab, ureB and cagA by other groups [22-24].

NifK gene is a key nitrogenase and plays an important role in the nitrogen fixation process. It is necessary to obtain the nifK gene for exploring its relationship with nitrogen fixation. This study aimed to clone nifK gene, predict the secondary structure, the characters and functions of the nifK protein, and conduct prokaryotic expression. According to other nifK sequences registered in the NCBI, specific primers were designed. Klebsiella variicola DX120E nifK gene opening reading frame (ORF) was cloned by PCR amplification, and its nucleotide sequence, amino acid sequence and protein structure were analyzed by combining bioinformatics methods. Prokaryotic expression vector pET30a (+) was used to construct the recombinant expression vector pET30a-nifK. After PCR, double enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21 (DE3), and 1.0 mmol/L IPTG was used to induce the gene expression at 28 o C. The fusion protein expression was

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expres- sion showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37 ℃ . SDS–PAGE analysis revealed that the ac- cumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and pu- rified by a 6 × His tagged affinity column under dena- turing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal anti- bodies, which were suitable to detect both the recom- binant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immuno- histochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on bio- logical function and application of FUS1.

Nucleoprotein of influenza virus, which has conservatory sequence and immunodominant epitopes being good CTL targets for releasing T-cell cross protections, is appropriate candidate for making universal vaccine (14). NP was used in different types of vaccines such as DNA vaccines (15), viral vector vaccines (16), subunit protein vaccines (17) and multi gene vaccines (18) which all of them show virus clearance and effective immunization in virus challenge infections. Also efficacy of NP in vaccine has been compared with other viral proteins, HA & M2, against subtypes of influenza virus and the results show good capacity of this protein in immunization but it needs combination to another molecule to enhance its efficacy (7). Prokaryotic expression of NP, especially in E. coli, in both wiled type and optimized formation is achievable (19, 20). NP derived from E. coli can be good candidate for vaccination and elicits robust cross-protection in mice(5).Following this results we could

Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Me- thods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps (lipo- polysaccharide), and we got two splicing products of mLRG (mLRGW, mLRGS) and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 (DE3). Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp re- spectively (mlrgW mlrgS). His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns; mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.

Unless otherwise indicated, standard molecular biol- ogy protocols were used according to Sambrook et  al. [30]. Restriction enzymes, Pfu polymerase and T4 ligase were from Promega (Madison, WI, USA). The coding sequence of dimeric C-terminal domain of human ZnT8 consisting of amino acids 268–369 [Arg325] and amino acids 268–369 [Trp325] were optimized for prokaryotic expression. The ZnT8 optimized nucleotide sequence was synthesized by GenScript (GenScript Corporation, Piscataway, NJ, USA; http://www.GenScript.com) includ- ing KpnI and XbaI sites at the 3 ′ and 5 ′ ends, respectively. The synthesized construct (682  bp) was obtained from GenScript in plasmid pUC57 and maintained in E. coli strain JM109 (Promega, Madison, WI, USA). After prop- agation and purification of the vector with QIAprep spin

parameters, its expression level is relative constancy in cells) were UL45 F (5’- CATGGAGTTGGGTGTGCT -3’) and UL45 R (5’-ACGCTGTAGTCGGTATCG -3’), b-actin F (5’-CCGGGCATCGCTGACA-3’) and b-actin R (5’- GGATTCATCATACTCCTGCTTGCT-3’). Stan- dard curve of PMD18-T/UL45 and PMD18-T/b-actin (constructed and preserved in the author ’ s laboratory) was established. The transcription kinetics of the DEV UL45 gene during the viral infection was detected by the method of FQ-PCR. The real-time FQ-PCR was per- formed in an 20 μ l reaction mixture containing cDNA 2 μ l, SYBR Green I Mix 9 μ l, each of the primer 25 pmo1, adding ultrapure water to total volume. Each run con- sisted of initial denaturation at 95°C for 1 min followed by 40 consecutive cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s. Then the fluorescence was measured by 94°C for 60 s and 60°C for 60 s, and then followed by 70 consecutive cycles of 60°C for 10 s, with each cycle increased 0.5°C. b-actin served as the internal parameters done the parallel experiment. Samples and internal parameters were tested in triplicate. The method of 2 - ΔΔ Ct was convenient to measure the relative amount of the UL45 mRNA expression [52].

Peptide transporter 2 is a high affinity and low capacity transporter, that has recently been found in the mammalian kidney, brain, lung, and mammary glands, which is known to transport dipeptides and tripeptides and to reabsorb small peptides generated by luminal peptidases in the kidney. In addition, peptide transporter 2 can transport peptide-like drugs and play important roles in the transshipment and positioning of drugs. In this paper, an E.coli BL21(DE3) containing pET30a(+)-hPepT2(560-663) recombinant plasmid was obtained by using PCR, double restriction enzyme digestion, ligation, and transformation. Then, the effect of changing both the induction time and induction dose using isopropyl β-D-1-thiogalactopyranoside on the bacterial expression of hPepT2(560-663) was detected by SDS-PAGE electrophoresis, the optimum expression conditions (at 37°) were identified as: 3 h and a working concentration of 0.5 mM isopropyl β-D-1-thiogalactopyranoside. Finally, we purified the target protein hPepT2(560-663) via Sephadex G-75 gel chromatography. These results will facilitate in future studies on the nature and function of the protein.

To produce parvovirus B19 antigen for diagnostic purposes, partially overlapping segments covering the genes encoding the viral structural proteins VP1 and VP2 were cloned into expressio[r]

We isolated the virus (M strain, the same below) from Aedes albopictus in the nature of Ma Wei town, Dushan County, Guizhou Province of Aedes albopictus in 2002. The virus was identi- fied as DENV-2 by indirect immunofluorescence with anti DENV1-4 monoclonal antibodies and digested genotyping of RT-PCR products. Compared with DENV-2 NGC strain, there are a base insertion and five mutations in the virus sequence. The virus sequence was recorded by GenBank, and the number is AY278226 [11, 18]. The sequence studied in the present work is 1-476 bp of DENV-2 E gene, a core area formed by folded monomer [19]. Our targeted sequence located in E protein EDI domain, an important domain involved in by protein E medi- ating membrane fusion process [15, 20]. Based on the work described as above, we pre- pared fusion protein of E gene 1-476 sequence of DENV-2 M strain and NGC strain, named M476 and N476 respectively. We used the pro- karyotic expression system pET28a+ to express protein, purified the protein with the affinity arm of the 6xHis-tag at the N or C-terminus of the target protein. To explore the effect of DENV E recombinant protein on the permeability of cultured human umbilical vein endothelial cell (HUVEC), polyclonal antibody of M-476 and N-476 were prepared. Our probe of the effect of protein E on the vascular permeability would benefit the understanding of the mechanism of DHF/DSS development.

Abstract: Objective: To express and purify the Toxoplasma gondii RH strain immune mapped protein 1 (IMP1) pro- tein through the prokaryotic expression system, and to identify the purified protein. Method: Gene of TgIMP1 was attained by RT-PCR and the amplified product was identified by TA-clone and sequencing. The identified fragment of IMP1 was connected into the expression vector pET28b and the recombinant pET28b-TgIMP1 was identified by double digestion and sequencing. The fusion protein TgIMP1 with 6 × His tag was expressed in E. coli BL21 (DE3) host and the optimal inducing conditions were investigated by adjusting the harvesting time interval, inducing temperature and concentration of isopropyl β-D-thiogalactoside (IPTG). The solubility detection of TgIMP1 protein and the massive purification of it were conducted with Ni 2+ -affinity purification. The purified product was identified

This study compared the suitability of four different expression systems for the production of CDH. We eval- uated the titer of secreted CDH, the purification proce- dure and yield together with the molecular and kinetic properties of the purified, recombinant enzymes. The prokaryotic expression host E. coli can express only the DH domain of CtCDH, but produced a high amount of it in a much shorter time (28 h) than the tested eukaryotic expression systems. Per liter of fermentation medium 652 U of CDH activity were obtained, which is 1.7-fold higher than the previously reported amount of the DH domain from Phanerochaete chrysosporium (PcDH) expressed in E. coli [18].

By using double antibody sandwich method to establish antigen capture ELISA method have very strict requirements of antibody specificity, and obtain specific antibodies requires high purity of immune antigen. Because of the H9 subtype avian influenza virus antigen preparation process is complex, lack of standardization, not easy to purify, and there is a risk of the spread of the virus, so get the artificial antigen specific H9 subtype avian influenza viral protein by using gene engineering technology has gradual importance. Because the HA protein is AIV specific, protective antigen, the antibody level of HA protein produced with whole virus antigen antibody level were almost consistency. For the expression of HA protein, it is important to choose the expression system, the Escherichia coli expression system has the advantages of low cost, convenient, simple operation, cultivating a large quantity, and pET30a prokaryotic expression vector expression of the N-terminal His tag which can be purified by affinity chromatography, the purity of nickel ions, can guarantee the maximum possible recombinant protein the purification process, simple, easy to mass production. In view of this, this study uses the pET30a prokaryotic expression vector in BL21 (DE3) to obtain the recombinant protein with high purity, the recombinant proteins were prepared highly specific monoclonal antibodies and polyclonal antibody. This study will be creative polyclonal and monoclonal antibodies were used as the capture antibody and detection antibody, polyclonal antisera because as capture antibody, has a wide range of capture sample, and the detection of antibody against H9 was used AIV HA protein specific monoclonal antibody, has an extremely high subtype specific, so the research establishment antigen capture ELISA method has the repeatability, sensitivity and specificity of good, and has the advantages of rapid, simple, economical, safe, high throughput, this method still needs further optimization and improvement of this method, firstly, the need for sample detection amount, accurately to determine the positive detection rate and false positive rate; secondly, the method can also be compared with the commercial ELISA kit or other ELISA detection method, in order to make the method more perfect, promotion and a wider range of applications.

The host immune response to Schistosoma japonicum is similar to that to S. mansoni infection, typically featuring Th2 polarization after egg laying. The major immune regulatory molecules of Omega-1 and IPSE in S. mansoni eggs have not been found in S. japonicum eggs. Therefore, the S. japonicum egg antigens inducing Th2 polarized re- sponses and related mechanisms are still poorly under- stood. Our group previously screened a batch of genes encoding secreted proteins with a signal sequence from a S. japonicum egg cDNA library by the Signal Sequence Trapping (SST) method [33]. Among them, the Sj CP1412 protein was found by amino acid sequence alignment to contain the conserved functional domains (CAS-1 and CAS-2) of the ribonuclease T2 family. Although the hom- ology between amino acid sequences of the Sm Omega-1 and Sj CP1412 molecules is low, Sj CP1412 was hypothe- sized to be another novel member of the ribonuclease T2 family in schistosomes. In the current study, the recom- binant Sj CP1412 protein (rSj CP1412) was prepared by a prokaryotic expression system and demonstrated to have ribonuclease activity, which could inhibit DC cell activa- tion, promote M2 macrophage differentiation, increase the number of Treg cells and drive the Th2 polarization of the host immune response. These findings support Sj CP1412 as an important immune regulatory factor in S. japonicum eggs.

Materials and Methods: Total RNA was extracted from MA104 cells infected with bovine rotavirus strain RF. BRV VP7 gene was amplified using polymerase chain reaction. Another gene was the VP7 synthetic that made in the pBSK plasmid. The pBSK-VP7 plasmid was digested using BamHI and SacI restriction endonucleases, then recombined into the prokaryotic expression vector pET32a. The pET32a-VP7 amplified and pET32a-VP7 synthetic were transformed into BL21 (DE3) competent cells of Escherichia coli, respectively, and induced by different concentrations of Isopropyl β-D-1-thiogalactopyranoside at 30°C and 37°C in luria bertani and terrific broth media, then analyzed using SDS-PAGE and western blotting. Results: SDS-PAGE analysis showed that the both pET32a-VP7 amplified and pET32a-VP7 synthetic were not expressed in the BL21 (DE3) cells, But the expression of pET32a-VP7 synthetic was weak at 30° C in luria bertani media. Conclusion: This is the first report of the production of bovine rotavirus (RF strain) Full-Length VP7 in prokaryotic system expression. VP7 is a membrane protein and has toxic domains that show high toxicity in this expression system.

Materials and Methods: We present here cloning and expression of influenza A virus M2 protein as a fusion with 6-His tag in Escherichia coli BL21 strain. The gene was amplified by PCR and ligated into the prokaryotic expression vector pET28a. The expression of M2 protein was induced by IPTG and confirmed by SDS-PAGE and western blotting. The desired protein was purified with affinity chromatography on a Ni-TED resin column and has to be evaluated in animal models for further studies.

prokaryotic expression of the candidate gene is usually necessary to prepare antiserum for verification of the antigenicity of the candidate gene or to prepare antigen for monitoring the efficiency of the DNA vaccine (eukar- yotic expression vector of the target gene). The introduc- tion of prokaryotic expression elements and the precise design of the compatible RBS and Kozak sequence endow pOmni with pro/eukaryotic dual-expression capabil- ity, which was validated by cloning and expression of reporter gene EGFP in E. coli and HeLa cells in this study. pOmni has two technical advantages compared with the only pro/eukaryotic dual-expression vector pDual GC (Agilent, USA) that we know. (1) pOmni is much smaller in size (3.7 vs. 6.6 kb) that means higher cloning capabil- ity for large DNA fragments. (2) pOmni has selection- free PCRRC function as discussed above, but with pDual GC, a target gene has to be cloned by means of traditional and troublesome restriction-mediated cloning method.

four different methods. First, we found no other mutations in the coding region of the CBS gene, which could be responsible for the severely elevated homocysteine and methionine levels. Second, the mutation was absent in 80 control alleles, indicat- ing that it is not a benign polymorphism. Third, the wild-type aspartic acid-444 residue is conserved in the evolution, which is evidence for functional relevance of this amino acid in the bio- logical function of the protein. Fourth, we observed a defective regulation of CBS by AdoMet in extracts of cultured fibro- blasts of this patient and in a prokaryotic expression system in which we introduced this mutation.

ABSTRACT Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition—and sometimes cleavage— of complementary targets. While eukaryotic Ago (eAgo) pro- teins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) pro- teins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are in- volved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing.

As mentioned above, kaiC appears to be an internally duplicated version of a recA/dnaB-like gene; it has two parts that are very similar to each other (Iwasaki et al., 1999). In each half of the kaiC gene, there is a Walker A or “P-loop” motif. This motif, [G or A]XXXXGK[T or S], is often a ATP/ GTP nucleotide binding region. Indeed, KaiC binds ATP, and when the Walker A motifs are mutated, the nucleotide binding is eliminated and rhythmicity is severely disrupted or abolished (Nishiwaki et al., 2000; Mori and Johnson, 2001b). Therefore, this motif is essential in both parts of the KaiC molecule for rhythmicity. As with eukaryotic clock proteins, interactions are important. KaiA, KaiB, and KaiC interact with each other (Iwasaki et al., 1999: Xu et al., 1999; Taniguchi et al., 2001). KaiC also interacts with a histidine kinase, SasA, that is crucial for maintaining a high amplitude, robust rhythm of gene expression (Iwasaki et al., 2000). These interactions appear to lead to the formation of complexes in vivo (Kageyama et al., 2003). There is also evidence for the association of a population of KaiB and KaiC molecules with membranes (Kitayama et al., 2003).