OBJECTIVETo identify the interaction partners of a new splicing product of LMO2 gene (LMO2-C), and study its function in K562 cells.

METHODSMaltose binding protein (MBP) pull down and mammalian two-hybrid assay (MTHA) were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein (GPA) in K562 cells.

RESULTSMBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pull down analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being (81.13 +/- 0.68)%. RT-PCR results showed that the expression level of GPA was reduced [(51.00 +/- 1.58)%] in K562 cells while LMO2-C overexpressed.

CONCLUSIONLMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.

Adaptor Proteins, Signal Transducing ; DNA-Binding Proteins ; genetics ; metabolism ; GATA1 Transcription Factor ; metabolism ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; genetics ; metabolism ; Periplasmic Binding Proteins ; Proto-Oncogene Proteins ; RNA Splicing ; Transcription Factors ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo Investigate the differences of sorbitol fermentation related genes and optimize molecular analysis method for distinguishing an epidemic with nonepidemic strains of Vibrio cholerae.

METHODSSequence analysis on four genes of sugar fermentation stimulation protein, periplasmic maltose-binding protein, periplasmic phosphate-binding protein and periplasmic amino acid-binding protein.

RESULTSIn this study, the following data was noticed: for O1 serogroup El Tor biotype V. cholerae, twenty-four epidemic and eight nonepidemic strains were chosen; For O139 serogroup V. cholerae, five epidemic and four nonepidemic strains were chosen. With those genes of sugar fermentation stimulation protein, there were three point mutations. The 106th, 150th, 378th oligonucleotide in epidemic strains were A, A and T, comparing to the nonepidemic strains which were G, G and C. When comparing the protein sequences, epidemic strains had a Threonine at 36th amino acid, whereas nonepidemic strains had an Alanine. The results in O139 serogroup were consistent with those in O1 serogroup El Tor biotype strains. Another two point mutations were found in the genes of periplasmic maltose-binding protein. The 999th, 1003rd oligonucleotides in epidemic strains were A and C, while in nonepidemic which were G and T. For the gene of periplasmic amino acid-binding protein, two point mutations were noticed. The 504th and 690th oligonucleotides in epidemic strains were T and C, but were C and T in nonepidemic. However, no amino acid differences were found in periplasmic maltose-binding protein and periplasmic amino acid-binding protein. For periplasmic amino acid-binding protein gene, there was no difference on oligonucleotide between epidemic and nonepidemic strains.

CONCLUSIONResults suggested that SNPs in these genes might serve as a useful tool to distinguish the epidemic strains from nonepidemic strains. The 36th amino acid mutation of sugar fermentation stimulation protein in epidemic and nonepidemic strains might change the activity of the protein which might be associated with sorbitol fermentation.

Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; Carrier Proteins ; genetics ; metabolism ; Fermentation ; Maltose-Binding Proteins ; Molecular Sequence Data ; Periplasmic Binding Proteins ; genetics ; metabolism ; Phosphate-Binding Proteins ; genetics ; metabolism ; Point Mutation ; Sequence Analysis, Protein ; Sorbitol ; Vibrio cholerae ; genetics ; metabolism

Animals ; Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Brucella Vaccine ; immunology ; Brucella abortus ; immunology ; Female ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Periplasmic Binding Proteins ; genetics ; immunology ; Ribosomal Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology

The formation of disulfide bonds in secreted proteins of E. coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG. DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it. Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates. DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo. It is regarded that DsbE has the similar function with DsbD. All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds. Furthermore, their chaperone activity can promote the formation of protein disulfide bonds. There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E. coli. So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG. Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E. coli, and DsbG has additive effects with DsbC.
Bacterial Proteins ; chemistry ; physiology ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Genetic Engineering ; methods ; Membrane Proteins ; chemistry ; physiology ; Molecular Chaperones ; physiology ; Oxidoreductases ; chemistry ; physiology ; Periplasmic Proteins ; Protein Disulfide-Isomerases ; chemistry ; physiology ; Recombinant Proteins ; biosynthesis

The robust capacity of skeletal muscle stem cells (SkMSCs, or satellite cells) to regenerate into new muscles in vivo has offered promising therapeutic options for the treatment of degenerative muscle diseases. However, the practical use of SkMSCs to treat muscle diseases is limited, owing to their inability to expand in vitro under defined cultivation conditions without loss of engraftment efficiency. To develop an optimal cultivation condition for SkMSCs, we investigated the behavior of SkMSCs on synthetic maltose-binding protein (MBP)-fibroblast growth factor 2 (FGF2)-immobilized matrix in vitro. We found that the chemically well-defined, xeno-free MBP-FGF2-immobilized matrix effectively supports SkMSC growth without reducing their differentiation potential in vitro. Our data highlights the possible application of the MBP-FGF2 matrix for SkMSC expansion in vitro.
In Vitro Techniques ; Maltose-Binding Proteins ; Muscle, Skeletal ; Muscles ; Stem Cells

The crystalline surface layer protein (SLP) and a 28-32 kDa antigen of Rickettsia typhi were known as strong immunogens. We previously reported a cloning and sequence analysis of the SLP gene of R. typhi (slpT) and showed that the open reading frame of this gene encodes both the SLP and a 32-kDa protein. Our study also showed that a 48-kDa protein reacted strongly with polyclonal antiserum of a patient with murine typhus. In this study, we produced three recombinant proteins (SLP, 32-kDa, and 48-kDa protein) in E. coli as fusion proteins with maltose binding proteins. The reactivity of these proteins with patients' sera was investigated.
Clone Cells* ; Cloning, Organism* ; Crystallins ; Humans ; Maltose-Binding Proteins ; Open Reading Frames ; Recombinant Proteins ; Rickettsia typhi* ; Rickettsia* ; Sequence Analysis ; Typhus, Endemic Flea-Borne

OBJECTIVETo economically obtain the Abeta peptide for Alzheimer's disease (AD) research by expressing the Abeta peptide fused with the maltose binding protein (MBP) possessing high solubility in E.coli.

METHODSThe cDNA-coding sequence of Abeta peptide was modified by the addition of a BamH I site at the 5' end and a Hind III site at the 3' end using PCR. The modified sequence was ligated into the maltose-binding protein (MBP) fusion expression vector pMAL-c2 containing an thrombin cleavage site, which was transformed into competent E.coli DH5alpha cells. After identification of the single clones by PCR and DNA sequencing, the recombinant plasmid was transformed into E.coli TB1 and induced to express MBP-Abeta fusion protein. The expressed fusion protein was purified using amylose resin column and identified by SDS-PAGE and Western blotting.

RESULTSThe result of DNA sequencing verified the consistency between the inserted sequence and Abeta (1-42) sequence. SDS-PAGE electrophoresis showed that MBP-Abeta fusion protein was highly expressed in E.coli TB1, and Western blotting demonstrated that the purified fusion protein and the separated Abeta peptide could be recognized by specific anti-Abeta (22-35) antibody.

CONCLUSIONMBP-Abeta fusion protein highly expressed in E. coli TB1 cells with enhanced solubility and the separated Abeta peptide with good immunogenicity obtained may lend support to AD research.

Alzheimer Disease ; genetics ; Amyloid beta-Peptides ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Maltose-Binding Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.
Animals ; Antibodies, Monoclonal* ; Bacteriophage T7 ; Blotting, Western ; Clone Cells ; COS Cells ; Cytoplasm ; Escherichia coli ; Fluorescent Antibody Technique ; Humans* ; Immunization ; Maltose-Binding Proteins ; Mice ; Microscopy, Electron

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
Bacterial Proteins ; biosynthesis ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; D-Amino-Acid Oxidase ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics ; Yeasts ; enzymology ; genetics

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism