1.Studies on expression, purification, crystal growth and optimization of putative transcription factor LytR from Streptococcus pneumoniae.
Xun MIN ; Wen ZHONG ; Shasha ZHAO ; Jie DONG ; Shanshan DONG ; Aie ZHOU ; Wenjuan YAN ; Deqiang WANG
Journal of Biomedical Engineering 2013;30(4):812-821
The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
Bacterial Proteins
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biosynthesis
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genetics
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isolation & purification
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Escherichia coli
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genetics
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metabolism
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Recombinant Proteins
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biosynthesis
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genetics
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isolation & purification
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Streptococcus pneumoniae
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genetics
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metabolism
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Transcription Factors
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biosynthesis
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genetics
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isolation & purification
2.Prokaryotic expression and purification of SPAG4L, a novel human testis gene.
Xian-zhen JIANG ; Ming-gang YANG ; Xiao-wei XING
Journal of Southern Medical University 2010;30(9):2047-2050
OBJECTIVETo express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.
METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.
RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.
CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.
Carrier Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Humans ; Male ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification
3.De novo design, non-chromatographic purification and salt-effect of elastin-like polypeptides.
Kaizong HUANG ; Jingjing LI ; Wei LI ; Huihua GE ; Wenyan WANG ; Guangya ZHANG
Chinese Journal of Biotechnology 2011;27(4):653-658
Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl beta-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 degrees C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.
Chromatography
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Elastin
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biosynthesis
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genetics
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isolation & purification
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Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Peptides
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genetics
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isolation & purification
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metabolism
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Recombinant Proteins
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biosynthesis
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genetics
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isolation & purification
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Sodium Chloride
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pharmacology
4.Expression and purification of recombinant metallothionein of Eriocheir sinensis in E. coli.
Xiaomin CAO ; Bing LI ; Zhen CHEN ; Hongbin TU ; Xin FU
Journal of Biomedical Engineering 2007;24(2):409-419
Metallothioneins (MT) are potential candidates for medicine development and application. For the purpose of expressing recombinant MT in E. coli, a crab MT cDNA cloned into pGEM-T was subcloned into pET-GST and then transformed into Escherichia Coli BL21. The fusion protein was proved to be expressed in both soluble and insoluble form by SDS-PAGE and western blot. Since metallothionein chelate metal ions, which may effects the physiological process of E. coli, caused the production of recombinant protein was lower than expected. Optimization of the ions content in the culture medium improved expression. The protein was purified by Zn2+ affinity chromatography, and rinsed off with high imidazole (1.5 M) which was the result of MT chelating instead of His-tag. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.
Animals
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Brachyura
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genetics
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Escherichia coli
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genetics
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metabolism
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Metallothionein
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biosynthesis
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genetics
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isolation & purification
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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isolation & purification
5.Optimization of plant des-pGlu1-Brazzein gene according to yeasty biased codons and its expression in Pichia pastoris.
Chunli LI ; Lu HAN ; Zhenyu ZHENG ; Weidong ZHAO
Chinese Journal of Biotechnology 2011;27(8):1158-1163
According to the amino acid sequence of des-pGlu1-Brazzein, 4 pairs of oligonucleotide with cosmic site were synthesized by using yeasty biased codons. After linkage and PCR, the 179 bp code area of des-pGlu1-Brazzein was obtained and inserted into pPIC9K, which resulted in the recombinant expression vector pPIC9K-Bra. By digestion with Sal I, the lined pPIC9K-Bra was transformed into Pichia pastoris GS115 by electric shock. The results of expression indicted that the secreted target protein accounted for 51.6% of total protein in the supernatant and showed biological activity after purification.
Codon
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Pichia
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genetics
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metabolism
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Plant Proteins
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biosynthesis
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genetics
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Recombinant Proteins
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biosynthesis
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genetics
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isolation & purification
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Sweetening Agents
6.Gene optimization is necessary to express HPV type 6 L1 protein in the methylotrophic yeast Pichia pastoris.
Ping-chuan LI ; Xiao-guang ZHANG ; Ling ZHOU ; Yi ZENG
Chinese Journal of Experimental and Clinical Virology 2003;17(4):310-314
OBJECTIVEHuman papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.
METHODSThe whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.
RESULTSThree clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.
CONCLUSIONHPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.
Capsid Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Genes ; Papillomaviridae ; genetics ; Pichia ; genetics ; metabolism ; Viral Proteins
7.Expression of acidophilic alpha-amylase from Alicyclobacillus acidocaldarius.
Tie-Zheng YUAN ; Bin YAO ; Hui-Ying LUO ; Ya-Ru WANG ; Ning-Feng WU ; Yun-Liu FAN
Chinese Journal of Biotechnology 2005;21(1):78-83
The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.
Bacillus
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enzymology
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genetics
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Bacterial Proteins
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genetics
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isolation & purification
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metabolism
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Cloning, Molecular
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Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Pichia
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genetics
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metabolism
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alpha-Amylases
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genetics
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isolation & purification
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metabolism
8.The preparation and identification of diagnostic recombinant glycerol kinase.
Yao MENG ; Zhenwei WANG ; Shuaikun WANG ; Jieqing HAO ; Hui SHI ; Yanfa MENG ; Shuangfeng LIN
Journal of Biomedical Engineering 2013;30(2):327-337
In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.
Escherichia coli
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genetics
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metabolism
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Fermentation
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Glycerol Kinase
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biosynthesis
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genetics
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isolation & purification
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Recombinant Proteins
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biosynthesis
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genetics
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isolation & purification
9.Expression, purification and characterization of bacteriophage lysin of Streptococcus in Escherichia coli.
Weiqing CHEN ; Xiaofeng WANG ; Pu WANG ; Deyong ZHANG ; Hong CHEN ; Wei KE ; Yin LU ; Jianfen ZHANG
Chinese Journal of Biotechnology 2009;25(8):1267-1272
Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis
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Enzymes
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biosynthesis
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genetics
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isolation & purification
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Escherichia coli
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genetics
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metabolism
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Recombinant Proteins
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biosynthesis
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genetics
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isolation & purification
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Streptococcus pyogenes
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drug effects
10.Optimization of expression and purification of recombinant Salvia miltiorrhiza WRKY1 protein in Escherichia coli.
Yu-Zhong LIU ; Ye SHEN ; Qi-Xian RONG ; Wen-Yan WU ; Rui-Bo LI ; Zhi-Gang WU ; Min CHEN
China Journal of Chinese Materia Medica 2014;39(7):1214-1219
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
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Cloning, Molecular
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DNA-Binding Proteins
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chemistry
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genetics
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isolation & purification
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metabolism
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli
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chemistry
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genetics
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metabolism
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Molecular Weight
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Plant Proteins
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chemistry
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genetics
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isolation & purification
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metabolism
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Recombinant Proteins
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chemistry
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genetics
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isolation & purification
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metabolism
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Salvia miltiorrhiza
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genetics