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Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

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Chemical Research in Chinese Universities 2008年第3期24卷 344-347页

作者： CHEN Yang YANG Su-juan fu Yao WANG Jia-peng ZHAO Zhi-zhuang fu xue-qi Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Jiltin University Changchun 130012 P R. China University of Oklahoma Health Science Centel Oklahoma 73104 USA

A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPα.

关键词： Receptor-like protein tyrosine phosphatase Clone Polyclonal antibodies

Expression and Characterization of the Intracellular Part of Receptor Protein Tyrosine Phosphatase PTPRU

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Chemical Research in Chinese Universities 2011年第3期27卷 474-477页

作者： WANG Shao-feng HAN Jia-yu fu xue-qi ZHAO Zhi zhuang Joe Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Jilin University Changehun 130012 P. R. China Department of Pathology University of Oklahoma Health Sciences Center Oklahoma City Oklahoma 73104 USA

PTPRU is an MAM domain-containing receptor-like protein tyrosine phosphatase. Previous studies have demonstrated an important role of the enzyme in the maintenance of epithelial integrity and in the regulation of the Wnt/β-catenin signaling pathway. To better understand the function of PTPRU, we cloned and expressed the intracellular portion of PTPRU as a GST fusion protein in E. coli cells. We purified the protein to homogeneity and used it to immunize mice for antibody production. The resultant antibody specifically recognized PTPRU over-expressed in the cell line. Western blot analyses demonstrated the partition of truncated forms of PTPRU containing the cadherin-like domain in the Triton X-100-insoluble fraction, and immunofluorescent cell staining revealed the localization of these proteins in punctate intracellular structures. Our data suggest that the cadherin-like domain of PTPRU has a major role in determining its intracellular localization.

关键词： Tyrosine phosphatase Cadherin Catenin Microdomain

Studies on HEPES Stabilizing M Intermediate of Bacteriorhodopsin

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Chemical Research in Chinese Universities 1995年第3期11卷 215-218页

作者： fu, xue-qi Li, Zheng-qiang Wang, Jin-dui Wang, Bao-hui Li, Bo-fu Li, Tie-jin JILIN UNIV NATL KEY LAB ENZYME ENGNCHANGCHUN 130023PEOPLES R CHINA JILIN UNIV KEY LAB MOLEC SPECTRUM & MOLEC STRUCTCHANGCHUN 130023PEOPLES R CHINA JILIN UNIV DEPT CHEMCHANGCHUN 130023PEOPLES R CHINA

EPES has proved to stabilize M intermediate of Bacteriorhodopsin. Absorp-tion and Raman spectra showed that there was a more stable form of M intermedi-ate in BR-HEPES solution. It was found by SPS that there were two... 详细信息

关键词： Bacteriorhodopsin M intermediate HEPES

Purification and Characterization of Protein Tyrosine Phosphatase MEG1 and Preparation of Anti-PTPMEG1 Antibody

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Chemical Research in Chinese Universities 2010年第4期26卷 591-595页

作者： ZHANG Xiao-ping XING Shu Xiao-xia LIN Fan fu xue-qi LI Wan-nan Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Jilin University Changchun 130012 P. R. China Clinical Laboratory China Japan Union Hospital Jilin University Changchun 130033 P. R. China Key Laboratory for Molecular Enzymology and Engineering Ministry of Education Jilin University Changchun 130012 P. R. China School of Life Science Northeast Normal University Changchun 130024 P. R. China

PTPMEGI is an intracellular protein tyrosine phosphatase（PTP）, which contains FERM and PDZ domains This study focuses our attention on the expression, purification and characterization of catalytic domain of PTPMEG1 （AMEG1） and preparation of its polyclonal antibody. A cDNA fragment encoding AMEG1 protein（amino acid residues 643-926） was amplified by PCR and then cloned into the pT7-7 vector. Both soluble and insoluble recombinant AMEG1 proteins were observed after induction by IPTG. Soluble AMEG1 was purified via two chromatographic steps, and the purified enzyme was characterized. With para-nitrophenylphosphate（pNPP） as a substrate, AMEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics. Insoluble AMEG1, which was mainly distributed in the inclusion body of E. coli cells extracts, was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody. A rabbit was immunized with AMEG1 purified by preparative electrophoresis to generate anti-AMEG1 antibody. Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography. The purified polyconal antibody displayed a satisfactory titer and sensitivity.

关键词： Protein tyrosine phosphatase(PTP) PTPMEG1 AMEG 1 Polyclonal antibody

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-ΔSHP-1 Antibodies

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Chemical Research in Chinese Universities 2008年第5期24卷 592-596页

作者： LI Wan-nan ZHUANG Yan LI He SUN Ying fu Yao WU Xiao-xia ZHAO Zhi-zhuang fu xue-qi Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Key Laboratory for Molecular Enzymology ＆ Engineering Ministry of Education dilin UniversilT Changchun 130021 P. R. China Department of Pathology The University of Oklahoma Health Science Center OK 73104 USA

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.

关键词： SHP-1 Protein tyrosine phosphatase Polyclonal antibodies

PRLs Promote Spreading, Adhesion, and Proliferation of Human SW480 and SW620 Cells

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Chemical Research in Chinese Universities 2006年第1期22卷 68-72页

作者： LI Zhao-fa XU xue-song SHEN Xing-gui LI qing-shan ZHAO Zhi-zhuang fu xue-qi LI Yu-lin LI Yi-lei Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Jilin University Changchun 130021 P. R. China China-Japan Union Hospital Jilin University Changchun 130031 P. R. China The Key Laboratory of Pathobiology Ministry of Education Jilin University Changchun 130023 P. R. China

Recent studies suggest that PRL-3 is involved in the metastasis of colorectal cancer, but the mechanism concerning that has not been well defined. This article expresses PRL-1, PRL-2, and PRL-3 and the catalytically inactive mutant forms of those enzymes in SW620 and SW480 cells, two human cell lines derived from non-metastatic cancer and metastatic colorectal cancer, respectively. While the expression of the native forms of PRLs promotes the spreading, adhesion, and proliferation of these cells, the expression of their mutant forms inhibits the earlier-mentioned processes. These data thus provide a cellular mechanism for the role of PRL-3 in tumor metastasis and suggest that all the three PRLs have similar functions.

关键词： PRL Metastasis Proliferation Expression SW480 cell SW620 cell

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

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Chemical Research in Chinese Universities 2011年第2期27卷 287-290页

作者： DONG Hong-bo LI Guo-dong WANG Shao-feng fu xue-qi ZHAO Zhi-zhuang Joe Edmond H. Fischer Signal Transduction Laboratory College of Life Science Jilin University Changchun 130012 P. R. China School of Pharmaceutical Science Jilin University Changchun 130021 P. R. China The Second Hospital of Jilin University. Changchun 130041 P. R. China Department of Pathology University of Oklahoma Health Sciences Center Oklahoma City Oklahoma 73104 USA

Protein tyrosine phosphatases（PTPs） are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting anti- serum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.

关键词： Protein tyrosine phosphatase Expression Antibody production Enzyme assay Tissue expression

SFNet:Stellar Feature Network with CWT for Stellar Spectra Recognition

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Research in Astronomy and Astrophysics 2024年第9期24卷 260-270页

作者： Hao fu Peng Liu Xuan qi xue Mei College of Electrical Engineering and Control Science Nanjing Tech UniversityNanjing 211816China

Stellar spectral classification is crucial in astronomical data ***,existing studies are often limited by the uneven distribution of stellar samples,posing challenges in practical *** when balancing stellar categories and their numbers,there is room for improvement in classification *** study introduces a Continuous Wavelet Transform using the Super Morlet wavelet to convert stellar spectra into wavelet images.A novel neural network,the Stellar Feature Network,is proposed for classifying these *** spectra from Large Sky Area Multi-Object Fiber Spectroscopic Telescope DR9,encompassing five equal categories(B,A,F,G,K),were *** experiments validate the effectiveness of the proposed methods and network,achieving significant improvements in classification accuracy.

关键词： methods data analysis-techniques image processing-stars imaging-galaxies stellar content

Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

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Chemical Research in Chinese Universities 2007年第6期23卷 697-700页

作者： SHEN Xing-gui LI Wan-nan WANG Jing JIANG Yi-qun LI qing-shan fu xue-qi Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Jilin University Changchun 130021 P. R. China

Phosphatase of regenerating liver 3 （ PRL3 ）, which belongs to the superfamily of protein tyrosine phosphatases （PTPs）, represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes. Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

关键词： PRL3 Prokaryotic expression Polyclonal antibody