Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.
Ebolavirus ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Nucleocapsid ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Virus Assembly

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.
Animal ; Baculoviridae/genetics ; Blotting, Western ; Cell Line ; Factor VIII/metabolism* ; Factor VIII/genetics ; Factor VIII/chemistry ; Factor VIII/biosynthesis ; Genetic Vectors ; Human ; Mutagenesis, Site-Directed ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/chemistry ; Recombinant Proteins/biosynthesis ; Spodoptera

We developed a new enzymatic-catalyzing producing process of glucose laurate monoester. In the process we used Candida antarctica lipase B-displaying Pichia pastoris whole-cells as biocatalyst, glucose as the acyl acceptor and lauric acid as the acyl donor. The product glucose laurate monoester was purified by silica gel column chromatography and preparative liquid chromatography, and identified by liquid chromatography-mass spectrometry. Then we optimized the process from various aspects, such as solvent composition, ratio of dmethyl sulfoxide to 2-Methyl-2-butanol (V/V), catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were: glucose 0.5 mmol/L, lauric acid 1.0 mmol/L, ratio of 2-Methyl-2-butanol to Dmethyl sulfoxide is 7:3 in 5 mL volume, temperature 60 degrees C, the best initial water activity of whole-cells biocatalyst is 0.11. The maximum glucose conversion could be 48.7% after 72 h.
Biocatalysis ; Candida ; enzymology ; Esters ; chemistry ; metabolism ; Fungal Proteins ; Genetic Engineering ; Glucose ; chemistry ; metabolism ; Laurates ; chemistry ; metabolism ; Lipase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. V_max, K_cat and K_m values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.
Cloning, Molecular ; Escherichia coli/metabolism* ; Phenylalanine/metabolism* ; Phenylalanine Ammonia-Lyase/chemistry* ; Recombinant Proteins ; Schisandra/genetics*

The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4.7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662 +/- 0.041) g/L, which is about 13.7 folds of the one optimized before.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Liver ; chemistry ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sharks ; metabolism

Halohydrin dehalogenase is of great significance for biodegradation of the chlorinated pollutants, and also serves as an important biocatalyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase (HheTM) gene from Tistrella mobilis KA081020-065 was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA column and characterized. Gel filtration and SDS-PAGE analysis showed that the native form of HheTM was a tetramer. It exhibited the highest activity at 50 degrees C. The nature and pH of the buffer had a great effect on its activity. The enzyme maintained high stability under the alkaline conditions and below 30 degrees C. HheTM catalyzed the transformation of ethyl(S)-4-chloro-3-hydroxybutyrate in the presence of cyanide, to give ethyl (R)-4-cyano-3-hydroxybutyrate, a key intermediate for the synthesis of atorvastatin.
3-Hydroxybutyric Acid ; chemistry ; Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; Hydrolases ; genetics ; metabolism ; Hydroxybutyrates ; chemistry ; Recombinant Proteins ; genetics ; metabolism ; Rhodospirillaceae ; enzymology ; genetics

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.
Animals ; Escherichia coli ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; HEK293 Cells ; Humans ; Insecta ; cytology ; genetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; metabolism ; Yeasts ; genetics

In order to express PebC1 in Pichia pastoris, the pebC1 sequence was amplified from genome Botrytis cinerea BC-4-2-2-1 by PCR and subcloned into the Pichua pastoris expression vector pPIC9K to generate pPIC9K-pebC1. The recombinant plasmid was linearized by Bgl II and transformed into Pichia pastoris GS115 by electroporation. Recombinant Pichia pastoris GS115/pPIC9K-pebC1 was screened by MD and G418-YPD plates and further confirmed by PCR. The protein expression was induced by methanol and analyzed by SDS-PAGE. SDS-PAGE analysis showed a special band about 39 kDa and western blotting indicated a good antigenicity of the expressed protein. Bioassay results showed that the recombinant protein PebC1 can induce resistance to gray mould disease of cucumber and Arabidopsi thaliana.
Botrytis ; chemistry ; Fungal Proteins ; biosynthesis ; genetics ; pharmacology ; Pichia ; genetics ; metabolism ; Plant Diseases ; prevention & control ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism

WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.
Blotting, Western ; Cloning, Molecular ; DNA-Binding Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; chemistry ; genetics ; metabolism ; Molecular Weight ; Plant Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Salvia miltiorrhiza ; genetics