OBJECTIVETo clone, express, and purify cyclic diadenosine monophosphate (c-di-AMP) metabolism-related genes from Porphyromonas gingivalis (P. gingivalis) ATCC33277.

METHODSPolymerase chain reaction (PCR) from the genome of P. gingivalis ATCC33277 amplified, the coding regions of pgn0523, pgn1187, and pgn2003 genes. The amplified DNA fragments were ligated with a prokaryotic expression vector pET28a to construct the recombinant expression plasmids pET-pgn0523, pET-pgn1187, and pET-pgn2003. These recombinant plasmids were transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells. The expression of recombinant proteins was induced by isopropyl-β-D-thiogalactoside and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were purified using a Ni²⁺ matrix column, and their concentrations were determined by a BCA Protein Quantitative Kit.

RESULTSThe c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 were amplified successfully with the correct molecular size. The recombinant expression vectors were constructed by ligating enzyme-digested PCR products and pET28a vector, and verified by PCR and sequencing. After induction and purification, recombinant proteins were expressed successfully and obtained with the correct molecular size (19.5 x 10³, 39.9 x 10³, 66.0 x 10³). The final protein concentrations were 0.708, 0.523, and 0.861 mg · mL⁻¹ after dialysis.

CONCLUSIONThe c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 are cloned successfully, and their coding products are expressed correctly in E. coli. High-purity proteins are finally obtained. The cloning and purification of these important proteins will help us to further investigate the physiological function and regulatory mechanism of c-di-AMP signaling system in P. gingivalis.

Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Dinucleoside Phosphates ; Escherichia coli ; genetics ; Genetic Vectors ; Plasmids ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; genetics ; Recombinant Proteins

OBJECTIVETo prepare the modified ZHER2V2 affibody with amino-terminal HEHEHE sequence and carboxyl-terminal GGGC sequence by gene recombinant expression,which is the basis for invasive HER2 imaging with affibody.

METHODSThe encoded affibody gene was optimized by codon preference of E. coli with gene designer software. The N-terminal of affibody was fused with HEHEHE sequence,while the C-terminal was fused with GGGC sequence. The synthetic gene was confirmed by Hind 3 endonuclease restriction and gene sequencing. The human epidermal growth factor receptor-2(HER2)affibody gene was sub-cloned into pET22b(+)plasmid and transformed into competent BL21(DE3)bacteria. The expression of modified affibody was induced with isopropyl Β-D-1-thiogalactopyranoside(IPTG)and identified by SDS-PAGE. The affibody was purified by nickel affinity binding and imidazole elution. The purified affibody was labeled with (68)Ga and its affinity was determined by saturation analysis with HER2-positive cells MDA-MB-361.

RESULTSThe affibody gene containing N-terminal HEHEHE and C-terminal GGGC sequences were confirmed by Hind 3 endonuclease restriction and gene sequencing. A newly expressed 8×10(3) protein was expressed from the induced recombinant bacteria identified by SDS-PAGE after sub-cloning HER2 affibody gene into pET22b(+)plasmid,transforming recombinant plasmid into competent BL21(DE3)bacteria and inducing the recombinant bacteria with IPTG. The expressed protein was purified from nickel agarose by 60 mmol/L imidazole eluting. The affinity Kd value of (68)Ga labeled affibody to HER2 positive MDA-MB-361 cells was 1.5 nmol/L.

CONCLUSIONThe affiibody ZHER2V2 containing N-terminal HEHEHE and C-terminal GGGC was successfully prepared by gene optimization,recombinant expression and affinity purification.

Affinity Labels ; isolation & purification ; Escherichia coli ; metabolism ; Gene Expression ; Humans ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; biosynthesis

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.
Adult ; Aged ; Antibodies, Viral/*blood ; Antigens, Viral/genetics/immunology/metabolism ; Capsid Proteins/genetics/immunology/metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin A/blood ; Immunoglobulin G/blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Recombinant Proteins/biosynthesis/genetics/immunology ; Republic of Korea/epidemiology ; Rotavirus/isolation & purification/*metabolism ; Rotavirus Infections/*epidemiology/virology ; Seroepidemiologic Studies ; Time Factors ; Young Adult

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.
Amino Acid Sequence ; Base Sequence ; Bupleurum ; chemistry ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Glycosyltransferases ; genetics ; isolation & purification ; metabolism ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Open Reading Frames ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; Protein Structure, Secondary ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saponins ; biosynthesis

The aim of this study is to establish stable transfected cell lines which could produce SPAG4L protein labeled with Myc and His tags in vitro. The open reading frame (ORF) of human SPAG4L was amplified by PCR and the fragments were cloned into eukaryotic expression vector pcDNA3.1/myc-His(-)A. The recombined plasmids of pcDNA3.1/myc-His(-)A/SPAG4L were verified by sequencing and digestion with enzymes. Then, the recombined plasmids were introduced into HeLa cells and screened by G418. Western blotting was performed to detect the expression of SPAG4L and its tags in stable transfected cell lines. SPAG4L and its tags were expressed in the stable cell lines transfected with pcDNA3.1/myc-His(-)A/SPAG4L, but not in the control group. Further study showed that SPAG4L colocalized with the endoplasmic reticulum(ER) marker PDI by immunofluorescence. The stable transfected cell lines established in this study will provide a powerful tool for further studies such as co-immunoprecipitation and pull-down.
Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Female ; Genetic Vectors ; genetics ; HeLa Cells ; Histidine ; genetics ; Humans ; Male ; Proto-Oncogene Proteins c-myc ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.
Escherichia coli ; genetics ; metabolism ; Fermentation ; Glycerol Kinase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.
Amino Acid Sequence ; Catalysis ; Coloring Agents ; metabolism ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Peroxidases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Zymomonas ; enzymology

The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pneumoniae ; genetics ; metabolism ; Transcription Factors ; biosynthesis ; genetics ; isolation & purification

Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Humans ; Microbial Sensitivity Tests ; Muramidase ; biosynthesis ; genetics ; Peptides, Cyclic ; biosynthesis ; genetics ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Plant betaine aldehyde dehydrogenase (BADH) is a physiologically important enzyme in response to salt or drought stress. In this study, two BADH genes (PeBADH1 and PeBADH2) were cloned from Populus euphratica. Both PeBADH1 and PeBADH2 genes encode the proteins of 503 amino acid residues, with a calculated molecular mass of 54.93 kDa and 54.90 kDa, respectively. Reverse transcription PCR showed the divergence of expression pattern between the PeBADH1 and PeBADH2 genes in P. euphratica. The recombinant PeBADH1 and PeBADH2 proteins were overexpressed in E. coli, and purified by Ni-affinity chromatography. The PeBADH2 protein had 1.5-fold higher enzymatic activity towards the substrate aldehyde than PeBADH1 protein. The PeBADH1 protein revealed higher thermal stability than PeBADH2 protein. These results indicated obvious functional divergence between the PeBADH1 and PeBADH2 genes.
Amino Acid Sequence ; Betaine-Aldehyde Dehydrogenase ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; physiology ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; chemistry ; genetics ; Populus ; genetics ; Protein Isoforms ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Substrate Specificity