1.Fusion tags technology and their applications.
Yong-Jin LI ; Yuan-Yuan CHEN ; Li-Jun BI
Chinese Journal of Biotechnology 2006;22(4):523-527
Fusion tags are originally developed to facilitate the purification of recombinant protein from crude extracts. In recent years, the discovery of different tags and the development of fusion strategy make the function of fusion tags diversified. However, there was no a cure-all fusion tag for different applications. We here give an overview of fusion tag technology and the different applications of fusion tags, including the purification, detection and oriented immobilization of recombinant protein, the visualization of bioevent in vivo, the enhancement of the yield of protein, the improvement of the solubility and stability of the expressed protein.
Recombinant Proteins
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chemistry
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isolation & purification
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Solubility
2.Research on expression of somatomedin b domain of proteoglycan 4 and recombinant protein aggregation.
Lifang WANG ; Zhibo HAN ; Wenhu CHEN ; Peng DU ; Aihua SUN ; Ping YANG ; Hongguang ZHAO
Journal of Biomedical Engineering 2014;31(6):1319-1324
Recombinant protein SMB(PRG4) containing two Somatomedin B domains and a small amount of glycosylation of repetitive sequences of proteoglycan 4 was cloned according to PGR4 gene polymorphism. Mature purification process was established and recombinant protein SMB(PRG4), with high-level expression was purified. By using size-exclusion chromatogaraphy and dynamic light scattering, we found that the recombinant protein self-aggregate to dimeric form. Structure prediction and non-reducing electrophoresis revealed that SMB(PRG4), was a non-covalently bonded dimer.
Glycosylation
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Protein Multimerization
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Proteoglycans
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chemistry
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Recombinant Proteins
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chemistry
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Somatomedins
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chemistry
3.Recent progress in fusion enzyme design and applications.
Ziliang HUANG ; Chong ZHANG ; Xi WU ; Nan SU ; Xinhui XING
Chinese Journal of Biotechnology 2012;28(4):393-409
Engineering and redesign of enzymes are important to industrial biocatalysis. Fusion enzyme technology, based on fusion protein design, is frequently used in multifunctional enzyme construction and enzyme proximity control. Here, we reviewed the recent progress in molecular design strategy and application studies of fusion enzymes. The concept and features of fusion enzymes were introduced, followed by a systematical summary of the design strategy of fusion enzymes. In particular, the effects of different linker properties on fusion enzymes and their possible mechanisms were discussed. In addition, recent studies on fusion enzyme applications were also discussed. Finally, based on our own studies on fusion enzymes and the current research progress, the key problems in fusion enzyme technology and perspectives of this field were discussed.
Biocatalysis
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Biotechnology
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Enzymes
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chemistry
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Protein Engineering
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Recombinant Fusion Proteins
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chemistry
4.Processing and Modification of Recombinant Spider Silk Proteins.
Bin LIU ; Tao WANG ; Xiaobing LIU ; Yongen LUO
Journal of Biomedical Engineering 2015;32(4):933-939
Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein.
Animals
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Arthropod Proteins
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chemistry
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Protein Engineering
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Recombinant Proteins
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chemistry
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Silk
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chemistry
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Spiders
5.Current status of researches in the development of blood tissue engineering product.
Fengjuan LI ; Jinfeng WANG ; Chengmin YANG
Journal of Biomedical Engineering 2008;25(4):972-975
The term "blood substitutes" includes plasma substitutes and blood cell substitutes in the broad sense, but in its narrow sense, it means red blood cell (RBC) substitutes, platelet substitutes and white blood cell (WBC) substitutes. The RBC substitutes includes perfluorocarbon, hemoglobin-based and encapsuled substitutes. The hemoglobin-based substitutes which was widely researched in the world includes human hemoglobin-based, animal hemoglobin-based and gene recombined hemoglobin based substitutes. The function and immunology of WBC is very complicated, so it is rarely used in clinic. Nowadays the platelet substitutes pursued by the researches and developments includes mainly the liposome and collagenic fiber species substitutes.
Blood Substitutes
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chemical synthesis
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Fluorocarbons
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chemistry
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Hemoglobins
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chemistry
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Humans
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Recombinant Proteins
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chemistry
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Tissue Engineering
;
methods
6.Site-specific PEGylation of recombinant lysostaphin.
Hairong LU ; Yitao ZHANGI ; Qingshan HUANG
Chinese Journal of Biotechnology 2016;32(1):127-134
Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.
Amino Acid Substitution
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Lysostaphin
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chemistry
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Polyethylene Glycols
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chemistry
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Protein Engineering
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Recombinant Proteins
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chemistry
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Staphylococcus aureus
7.Hydroxyapatite nanoparticles enhance the efficacy of liposome-mediated gene-transfection into HepG2 cells and its mechanisms.
Gao-Peng LI ; Xiao-Ping CHEN ; Zhi-Yong HUANG
Chinese Journal of Oncology 2008;30(2):111-112
Cell Proliferation
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DNA
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chemistry
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Durapatite
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chemistry
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Hep G2 Cells
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Humans
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Liposomes
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chemistry
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Nanoparticles
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chemistry
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Particle Size
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Recombinant Proteins
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chemistry
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Transfection
8.Process Optimization of PEGylating Fused Protein of LL-37 and Interferon-α2a.
Journal of Biomedical Engineering 2015;32(6):1261-1266
PEGylating is an effective way for prolonging the half-time period and decreasing the immunogenicity of protein drugs. With experiments of single factor, it was proved that the optimal processes for PEGylating the fused protein of LL-37 and interferon (IFN)-α2a were: PEG molecular weight was 5,000, fused protein concentration was 0.6 mg/mL, the mole ratio of protein to mPEG₅₀₀₀-SS was 1:10, the reaction temperature was 4 °C, and the pH was 9.0, respectively. With orthogonal experiments, we proved that the influential order of 3 main factors is: the fused protein concentration > the mole ratio of protein and mPEG₅₀₀₀-SS > pH and the optimal conditions were the fused protein concentration as 0.6 mg/mL, the mole ratio of protein and mPEG₅₀₀₀-SS as 1:10, pH as 8.8. Under these optimal conditions, the average rate of PEGylated protein with 3 times parallel experiments was 86.98%. After PEGylated, the interferon activity and antimicrobial activity of fused protein could be remained higher than 58% and 97%, respectively.
Anti-Infective Agents
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chemistry
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Antimicrobial Cationic Peptides
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chemistry
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Interferon-alpha
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chemistry
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Polyethylene Glycols
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chemistry
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Recombinant Proteins
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chemistry
9.Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII.
Sang Hwan OH ; Mi Young LEE ; Dong Weon SONG
Experimental & Molecular Medicine 1999;31(2):95-100
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
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Baculoviridae/genetics
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Blotting, Western
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Cell Line
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Factor VIII/metabolism*
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Factor VIII/genetics
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Factor VIII/chemistry
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Factor VIII/biosynthesis
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Genetic Vectors
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Human
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Mutagenesis, Site-Directed
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Recombinant Proteins/metabolism
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Recombinant Proteins/genetics
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Recombinant Proteins/chemistry
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Recombinant Proteins/biosynthesis
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Spodoptera
10.Construction of spider silk functional platform via intein trans-splicing.
Senzhu LIN ; Gefei CHEN ; Qing MENG
Chinese Journal of Biotechnology 2016;32(12):1704-1714
To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.
Animals
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Inteins
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Protein Splicing
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Recombinant Proteins
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chemistry
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Silk
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chemistry
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Small Ubiquitin-Related Modifier Proteins
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chemistry
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Spiders
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Trans-Splicing