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 ; Pichia ; genetics ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sweetening Agents

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

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 ; Brachyura ; genetics ; Escherichia coli ; genetics ; metabolism ; Metallothionein ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo establish the expression and purification route for human amelogenin mature peptide in Escherichia coli and obtain the purified amelogenin (AMG) mature peptide.

METHODSRecombined plasmid pGEX-4T-1-AMG was transformed to Escherichia coli BL21. After expression, AMG was purified with glutathione S-transferase fusion protein purification system (GSTrapFF) column.

RESULTSSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization results showed that 45,000 GST-AMG fusing protein and 19,000 target AMG mature peptide were obtained successfully.

CONCLUSIONSpGEX-4T-1-AMG-BL21 system is used successfully to express and purify human AMG mature peptide.

Amelogenin ; biosynthesis ; genetics ; isolation & purification ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Glutathione Transferase ; genetics ; isolation & purification ; Humans ; Peptides ; genetics ; isolation & purification ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism

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 ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pneumoniae ; genetics ; metabolism ; Transcription Factors ; biosynthesis ; genetics ; isolation & purification

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 ; genetics ; metabolism ; Fermentation ; Glycerol Kinase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

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 ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects

Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.
Apoptosis ; drug effects ; Escherichia coli ; genetics ; Humans ; Leptin ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

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 ; Elastin ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptides ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sodium Chloride ; pharmacology

Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme.
Cloning, Molecular ; Fungal Proteins ; chemistry ; genetics ; isolation & purification ; Muramidase ; chemistry ; Plasmids ; Protein Folding ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Spectrometry, Fluorescence