OBJECTIVETo construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry.

METHODSMatrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques were used to identify 12 recombinant proteins (10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori). A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-ClinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FlexAnalysis softwares.

RESULTSModels of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities: RC=100%, mean CVA=98.7% (the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively) and PPV=95%. This model can be used to classify the bacteria and their recombinant, which only requires 3.7×103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks (9, 16, 9, and 14 for the four stages, respectively) was found in the various phase of recombinant bacteria.

CONCLUSIONMALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.

Bacterial Proteins ; analysis ; Cloning, Molecular ; Escherichia coli ; Mass Spectrometry ; Organisms, Genetically Modified ; Peptide Mapping ; Recombinant Proteins ; analysis

OBJECTIVETo purify recombinant nuclear protein of Hantaan virus.

METHODSThe recombinant plasmid was transformed into E.coli and induced by IPTG. The expressed protein is a fusion protein with GST and existed in inclusion bodies. The inclusion bodies were processed through denaturation and renaturation and were purified with Glutathione Sepharose 4B affinity chromatography column. Then the purified fusion protein and nuclear protein were examined by sandwich ELISA and Western blot.

RESULTSThe expressed fusion protein was separated from the mixture proteins by the first affinity chromatography. GST was cut from the purified fusion protein with thrombin. The nuclear protein was separated from GST by the second affinity chromatography. The crossed peak represents nuclear protein and the elute peak represents GST. The purified fusion protein and nuclear protein were single band by SDS-PAGE. Both of them had available antigen activity.

CONCLUSIONSPurification of nuclear protein of Hantaan virus with Glutathione Sepharose 4B affinity chromatography is an effective method.

Blotting, Western ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Hantaan virus ; Nuclear Proteins ; analysis ; isolation & purification ; Plasmids ; Recombinant Fusion Proteins ; analysis ; isolation & purification ; Viral Proteins ; analysis ; isolation & purification

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
Chromatography, Ion Exchange ; methods ; Disulfides ; chemistry ; Fibrinolytic Agents ; analysis ; chemistry ; Hirudins ; analysis ; chemistry ; Protein Refolding ; Recombinant Fusion Proteins ; analysis ; chemistry ; Recombinant Proteins ; analysis ; chemistry ; Tissue Plasminogen Activator ; analysis ; chemistry

Ethyl carbamate (EC) as a potential carcinogen commonly exists in traditional fermented foods. It is important eliminate urea that is the precursors of EC in many fermented foods, including Chinese Rice wine. On the basis of achieving high-level overexpression of food-grade ethanol-resistant acid urease, we studied the hydrolysis of urea and EC with the recombinant acid urease. Recombinant acid urease showed degraded urea in both the simulated system with ethanol and Chinese Rice wine (60 mg/L of urea was completely degraded within 25 h), indicating that the recombinant enzyme is suitable for the elimination of urea in Chinese Rice wine. Although recombinant acid urease also has degradation catalytic activity on EC, no obvious degradation of EC was observed. Further investigation results showed that the Km value for urea and EC of the recombinant acid urease was 0.7147 mmol/L and 41.32 mmol/L, respectively. The results provided theoretical foundation for realizing simultaneous degradation of urea and EC.
Oryza ; Recombinant Proteins ; metabolism ; Urea ; chemistry ; Urease ; metabolism ; Urethane ; chemistry ; Wine ; analysis

BACKGROUNDTo express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.

METHODSBovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His.Tag affinity chromatography, and it bioactivity was analyzed.

RESULTSCompared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.

CONCLUSIONSThe EKL gene from Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.

Animals ; Catalytic Domain ; genetics ; Cattle ; Cloning, Organism ; DNA, Complementary ; Enteropeptidase ; analysis ; genetics ; Recombinant Proteins

Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.
Electroporation ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; analysis ; biosynthesis ; genetics

This study aimed to obtain a recombinant human-source collagen for industrialization. First, based on the Gly-X-Y sequence of human type I collagen, we optimized the hydrophilic Gly-X-Y collagen peptide, designed the human collagen amino acid sequence and the corresponding nucleotide sequence. Next, the expression vector pPIC9K-COL was constructed via endonuclease digestion technology. We obtained an engineering strain of human-source collagen by electrotransforming Pichia pastoris, and then it was fermented, purified and identified. As a result, the expression level reached 4.5 g/L and the purity was over 95%. After amino acid N-terminal sequencing, molecular weight analysis, amino acid analysis and collagenase degradation test, we confirmed that the obtained collagen was consistent with designed primary structure of human-source collagen. After freeze-drying, we analyzed the collagen by scanning electron microscope and cell cytotoxicity, confirming that the collagen has porous fiber reticular structure and superior cytocompatibility. This indicates that human-source collagen has potential to be applied as biomedical material. In conclusion, we successfully obtained the expected human-source collagen and laid a foundation to its further application.
Amino Acid Sequence ; Biocompatible Materials ; Collagen ; analysis ; Freeze Drying ; Humans ; Pichia ; Recombinant Proteins

AIMN-Glycans in recombinant human erythropoietin (EPO) are essential to in vivo biological activity. This paper is to develop a method for mapping sialyated or asialyated N-glycans of EPO.

METHODSAt first, N-glycans linked to asparagines in glycoprotein EPO were released by peptide N-glycosidase F. To map asialyated N-glycans, sialic acid in N-glycans were removed by incubating N-glycans with sialidase. Oligosaccharides were labeled with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate (APTS), and all of the labeled oligosaccharides released from EPO were mapped by capillary gel electrophoresis with laser-induced fluorescence. The relationship between N-glycans and bioactivity of EPO was investigated on the basis of N-glycan mapping spectra.

RESULTSN-Glycans of seven different batches of EPO were mapped. Each sample was analysed twice, with and without sialidase treatment. The results showed that N-glycans of each sample were approximately the same. But when the expression vector was different, the types of N-glycans and their relative content were quite different. In case of asialyated N-glycan mapping, the retention time of each oligosaccharide delayed greatly, and most importantly, the resulted sialic acid peak can be used as a quantitative standard to determine sialic acid content in N-glycans of EPO. In addition, the difference of N-glycan mapping was observed when the in vivo biological activity of EPO was different.

CONCLUSIONThe approach in this article for determining N-glycan mapping can be applied to determine the source of EPO and the difference between each batch. It is also a suitable tool for routinely controlling the inner quality of EPO by coupled with peptide mapping.

Electrophoresis, Capillary ; methods ; Erythropoietin ; chemistry ; Fluorescence ; Lasers ; N-Acetylneuraminic Acid ; analysis ; Peptide Mapping ; Polysaccharides ; analysis ; chemistry ; Recombinant Proteins

Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity. Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed. DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG. The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L. The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90%. The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD. This value and amino acid analysis were consistent with those of theoretic calculation.
Amino Acid Sequence ; Amino Acids ; analysis ; Base Sequence ; Escherichia coli ; genetics ; Fibroins ; Molecular Sequence Data ; Molecular Weight ; Proteins ; analysis ; genetics ; isolation & purification ; Recombinant Proteins ; analysis ; isolation & purification

With the development of bio-technological drugs, drug immunogenicity evaluation has become key factor of clarifying safety and efficacy of these drugs. It has become the focus to establish a stable and reliable evaluation system. Due to the advantages such as continuous real-time monitoring, surface plasmon resonance (SPR) technology has been widely used in bio-technological drugs immunogenicity assessments. Our study applied this technology to detect anti-drug antibody (ADA) of a recombinant human anti-rabies monoclonal antibody NM57 in the sera of 48 volunteers admitted in phase I clinical trials. This method could satisfy the basic requirements of detection of ADA.
Antibodies, Anti-Idiotypic ; analysis ; blood ; immunology ; Antibodies, Monoclonal ; analysis ; blood ; immunology ; Antibodies, Viral ; analysis ; blood ; immunology ; Humans ; Rabies virus ; immunology ; Recombinant Proteins ; analysis ; blood ; immunology ; Surface Plasmon Resonance