• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.574-579
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• 2014

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

• Journal of Industrial Technology
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• v.36
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• pp.33-37
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• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• Korean Journal of Veterinary Research
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• v.45 no.2
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• pp.185-189
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• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Journal of the Korea Institute of Military Science and Technology
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• v.14 no.4
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• pp.710-718
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• 2011

Edema factor(EF) is a portion of anthrax toxin which produces edema when combined with protective antigen. This paper describes about technique for cloning, expression, purification and activity test of EF. Using the E. coli expression system, we could make recombinant EF protein although it's origin is Bacillus anthracis. And also we could culture massively and purify highly pure protein. Finally we confirm a enzyme activity of purified EF to increase intracellular cAMP level. Through establishing this technique, it can be possible to research about EF in depth and apply to expression and purification of many other protein in biology.

• BMB Reports
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• v.41 no.5
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• pp.404-407
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• 2008

A 345-bp gene that encodes human bone morphogenetic protein-2 (hBMP-2) has been synthesized. The codon usage of the resulting gene was modified to include those triplets that are utilized in highly expressed Escherichia coli genes. The hBMP-2 gene was efficiently expressed in E. coli as a soluble and active protein. Since the recombinant hBMP-2 was readily solublized, no further solublization steps were required throughout purification. No additional tagging residues were introduced into the synthetic hBMP-2 gene product. The developed synthetic gene is a promising approach for scaling-up the soluble expression of hBMP-2.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Bulletin of the Korean Chemical Society
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• v.28 no.9
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• pp.1549-1552
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• 2007

The cytoplasmic domain of syndecan-4, a type I transmembrane heparan sulfate proteoglycan, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF and NMR spectroscopy. The yield of the peptide was 3.0-1.5 mg per liter in LB or minimal medium, respectively. The recombinant expression and purification of this domain will enable us its structural and functional studies using multidimensional NMR spectroscopy.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.620-623
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• 2003

The delta sleep-inducing peptides (DSIP, Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu) is an important regulatory hormone, controlling hypothalamus and pituitary functions. In the current study, an expression system was designed for the rapid and economic expression oi recombinant DSIP for biophysical studies. Artificially synthesized oligonucleotides encoding DSIP were cloned into a pGEX-KG vector and expressed in E. coli (BL21). The recombinant GST-DSIP was then readily purified using a GST affinity column. To obtain intact DSIP from the GST-DSIP, thrombin cleavage and a CNBr reaction were successively carried out. The DSIP in the CNBr reaction mixture was subjected to RP-HPLC purification to yield 1.2 mg DSIP from a 1 liter culture of E. coli. Identification of the DSIP was peformed using MALDI-MS and an amino acid composition analysis.

• Journal of the Korean Magnetic Resonance Society
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• v.17 no.1
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• pp.30-39
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• 2013

Immortalization-upregulated protein-1 (IMUP-1) genes have been cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 gene is highly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. Previously, there were several problems for overexpression of IMUP-1 in bacterial expression systems including low solubility and aggregation due to unstructured property. To investigate the structural properties, it is necessary to obtain lots of pure and soluble proteins. Accordingly, the co-expression systems of bacterial chaperone proteins, GroEL-GroES, were used to increase solubility of IMUP-1. From the analysis of NMR and CD experiment data, it is suggested that the protein adopt typical the random coil properties in solution.