To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 μg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5-1 100 ng/mL, with recovery rates between 93%-107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.
Animals ; Humans ; Immunoassay ; Luminescence ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/isolation & purification* ; Procollagen/isolation & purification*

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.
Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; isolation & purification ; Interleukin-6 ; chemistry ; isolation & purification ; Peptide Fragments ; chemistry ; isolation & purification ; Protein Folding ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti-virus and anti-tumor vaccines. However the expression level of whole length gp96 was relatively low in E. coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment (560aa-751aa) of murine gp96 into the pGEX-6p-1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein.
Animals ; Antigens, Neoplasm ; genetics ; Blotting, Western ; Chromatography, Gel ; Cloning, Molecular ; Mice ; Peptide Fragments ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.
Amyloid beta-Peptides ; genetics ; immunology ; Antibody Specificity ; immunology ; Humans ; Peptide Fragments ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.
Angiogenesis Inhibitors/pharmacology* ; Angiogenesis Inhibitors/isolation & purification ; Angiogenesis Inhibitors/genetics* ; Animal ; Blotting, Western ; Cattle ; Cell Movement/drug effects ; Chick Embryo ; Chorion/pathology ; Chorion/drug effects ; Circular Dichroism ; Collagen/pharmacology* ; Collagen/isolation & purification ; Collagen/genetics* ; Electrophoresis, Polyacrylamide Gel ; Endothelium, Vascular/drug effects ; Endothelium, Vascular/cytology ; Escherichia coli/genetics* ; Fibroblast Growth Factor, Basic/pharmacology ; Mice ; Neovascularization, Physiologic/drug effects ; Peptide Fragments/pharmacology* ; Peptide Fragments/isolation & purification ; Peptide Fragments/genetics* ; Protein Folding ; Recombinant Proteins/pharmacology ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/genetics ; Solubility ; Yeasts/genetics

Edible bird's nest contains lots of glycoproteins. The glycosylation inhomogeneity for glycoprotein often results in wide range of molecular weight and the difficulty for protein separation and charaterization. In this paper, proteins in the edible bird's nest were extracted using multiple extractions, and then digested by PNgase F and trypsin. The digest mixture was separated with HPLC, and peptides were identified based on MS/MS data searching. The results indicated that the extracted proteins were amount to 79.7% of total protein in the edible bird's nest. More than 20 species of peptides in the digested mixture were identified. The sequences of these peptides showed similarity with some proteins from Swiss-prot. The research indicated that deglycosylation, tryptic digestion coupled with HPLC-MS/MS is a proper strategy for characterization of proteins in the edible bird's nest.
Amino Acid Sequence ; Animals ; Birds ; Chromatography, High Pressure Liquid ; Glycoproteins ; chemistry ; Mass Spectrometry ; Medicine, Chinese Traditional ; Peptide Fragments ; chemistry ; isolation & purification ; metabolism ; Proteolysis

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.
Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Coronavirus ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; isolation & purification ; Peptide Fragments ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.
Animals ; Antibodies, Bacterial ; genetics ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Chromatography, Affinity ; Diphtheria Toxin ; immunology ; Escherichia coli ; genetics ; Female ; Immunotoxins ; isolation & purification ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; immunology ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo observe the effect of human vascular endothelial growth factor (VEGF) fragment (3 approximately 4 exon) in E. coli on anti-angiogenesis.

METHODSThrough RT-PCR amplification, endonuclease cut and DNA sequence analysis identification, hVEGF fragment cDNA was inserted into E. coli expression vector pTrcHis2A. The prokaryotic expression plasmid pTrcHis2A/VEGF(3 approximately 4) was constructed and transformed into TOP10F.

RESULTSAfter 8hr isopropy-beta-D-thiogalactoside (IPTG) induction, VEGF fragment was expressed in 15% of total proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expressed protein was highly antigenic and specific. The VEGF fragment was further purified by affinity, which could inhibit HUVEC proliferation and neovascularization of the chick chorioallantoic membrane.

CONCLUSIONVEGF fragment is anti-angiogenetic, which may potentially be used in oncologico-biological targeting therapy.

Angiogenesis Inhibitors ; isolation & purification ; pharmacology ; Cloning, Molecular ; Endothelial Growth Factors ; genetics ; isolation & purification ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; drug effects ; Genetic Vectors ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; isolation & purification ; pharmacology ; Isopropyl Thiogalactoside ; pharmacology ; Lymphokines ; genetics ; isolation & purification ; pharmacology ; Peptide Fragments ; genetics ; isolation & purification ; pharmacology ; Plasmids ; genetics ; Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.
Anti-HIV Agents/pharmacology ; Drug Resistance, Viral/*genetics ; HIV Envelope Protein gp41/*genetics/metabolism/pharmacology ; HIV Infections/virology ; HIV-1/*genetics/isolation & purification/*metabolism ; Humans ; Peptide Fragments/pharmacology ; *Polymorphism, Genetic ; Protein Structure, Tertiary/genetics ; Republic of Korea ; Virus Internalization