1.Prokaryotic expression and purification of different truncated protein of Mayven.
Fang LIU ; Yingxiong WANG ; Xueqing LIU ; Junlin HE
Journal of Biomedical Engineering 2008;25(6):1401-1404
To understand the function of Mayven and investigate the pathogenesis of multiple sclerosis, the gene sequences of different truncated Mayven were amplified from the gene library of human brain. These truncated fragments, including fragment P1 (1-902 bp), fragment P2 (1-523 bp), fragment P3 (507-182 bp) and fragment P4 (887-1782 bp), were cloned into pGEX-4T-2 vector to construct recombinant plasmids. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The expressed proteins were detected by SDS-PAGE and Western blot, and were purified by GST purifying system. The results showed that recombinant express vectors of different truncated GST-Mayven were successfully constructed and were expressed in soluble form protein induced by IPTG. The fusion proteins have good reactivity to GST antibody. The construction of recombinant express vectors of different truncated GST-Mayven lays a basis for further function study on Mayven.
Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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Humans
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Microfilament Proteins
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genetics
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metabolism
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Multiple Sclerosis
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genetics
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Nerve Tissue Proteins
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genetics
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metabolism
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Recombinant Fusion Proteins
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genetics
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metabolism
2.Expression of Calponin-1 and Transgelin in human uterine smooth muscles in non-labor and labor situation.
Qian CHEN ; Yonghong GU ; Changju ZHOU ; Lingyu HU ; Changying PENG
Journal of Central South University(Medical Sciences) 2010;35(10):1073-1079
OBJECTIVE:
To investigate the expression of Calponin-1 and Transgenlin in the uterine smooth muscles during normal labor on-sets, and to evaluate their effect on initiating the normal labor.
METHODS:
A total of 14 uterine bodies and lower segments of human pregnancy were divided to a non-labor group (NIL) and a labor group(IL). Immunohistochemical technology and Western blot were used to determine the expression of Calponin-1 and Transgelin in the 2 groups.
RESULTS:
Immunohistochemical detection and Western blot showed that Calponin-1 protein in the uterine smooth muscle tissue of the body and the lower uterine segment of smooth muscle tissues had significant difference (P<0.05). The expression of Transgelin in the uterine body smooth muscle tissue in the IL was higher than that in the NIL(P<0.05). In the lower uterine segments of the smooth muscle, the expression of Transgelin was not significantly different in the 2 groups (P>0.05).
CONCLUSION
Calponin-1 of the uterine smooth muscle and Transgelin of the uterine body smooth muscle may involve in the regulation of uterine smooth muscle contractility, which is closely related to child birth launch.
Adult
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Calcium-Binding Proteins
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genetics
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metabolism
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Female
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Humans
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Labor Onset
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metabolism
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Microfilament Proteins
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genetics
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metabolism
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Muscle Proteins
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genetics
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metabolism
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Myometrium
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metabolism
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Pregnancy
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Uterine Contraction
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metabolism
4.Construction of adenoviral vector encoding Calponin-1 SiRNA and its effect on human myometrial cells.
Yong-hong GU ; Chang-ju ZHOU ; Ling-yu HU ; Qian CHEN ; Wei-she ZHANG
Chinese Journal of Pathology 2009;38(2):125-126
Adenoviridae
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genetics
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Calcium-Binding Proteins
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genetics
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metabolism
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Cells, Cultured
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Female
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Genetic Vectors
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Humans
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Microfilament Proteins
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genetics
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metabolism
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Myocytes, Smooth Muscle
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metabolism
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Myometrium
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cytology
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metabolism
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RNA, Small Interfering
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genetics
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Recombinant Fusion Proteins
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genetics
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metabolism
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Transfection
5.Expression and significances of FSCN1 and HGF in nasal inverted papilloma.
Linlin YUAN ; Weihua LOU ; Jianzhong SANG
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2012;26(8):339-342
OBJECTIVE:
To study the expressions of FSCN1 and HGF in nasal inverted papilloma (NIP) and explore their role in occurrence and development of this disease.
METHOD:
Immunohistochemical method was used to determine the expression of FSCN1 and HGF in 12 cases of chronic hypertrophic rhinitis, 40 cases of NIP and 14 cases of NIP with malignant transformation.
RESULT:
FSCN1 was expressed in 52.5% of NIP, 78.6% of NIP with malignant transformation and 8.3% of inferior turbinate of chronic hypertrophic rhinitis. Expression of FSCN1 was significantly higher in NIP and NIP with malignant transformation than in inferior turbinate (P<0.05). HGF was expressed in 85.7% of NIP with malignant transformation and 8.3% of inferior turbinate. Expression of HGF was significantly higher in NIP with malignant transformation than in inferior turbinate (P<0.05). HGF was expressed in 40.0% of NIP,which was higher than that of inferior turbinate. Expression of HGF was positively related to expression of FSCN1 in NIP and NIP with malignant transformation.
CONCLUSION
The abnormal expression of FSCN1 and HGF may be closely correlated with NIP and its malignant process. Analysis of FSCN1 and HGF expression in NIP may be useful in predicting malignant transformation.
Carrier Proteins
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genetics
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metabolism
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Hepatocyte Growth Factor
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genetics
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metabolism
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Humans
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Microfilament Proteins
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genetics
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metabolism
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Nasal Mucosa
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metabolism
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pathology
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Nose Neoplasms
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genetics
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metabolism
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pathology
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Papilloma, Inverted
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genetics
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metabolism
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pathology
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Turbinates
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metabolism
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pathology
6.The expression of fibrillin 1 in pathologic scars and its significance.
Fang-Fei NIE ; Qi WANG ; Ze-Lian QIN
Chinese Journal of Plastic Surgery 2008;24(5):339-342
OBJECTIVETo probe into the mechanism of fibrillin 1 in pathologic scar, by examining the expressions of fibrillin 1 and TGF-beta1 as well as their correlations in the tissues of keloid, hypertrophic scar and normal skin.
METHODSThe tissues of keloid, hypertrophic scar and normal skin were tested. RT-PCR was used to assess the mRNA expression levels of the aimed genes. The distribution of fibrillin 1 in scars and normal skin was examined by immunohistochemistry staining.
RESULTSThe mRNA level of fibrillin 1 in keloid (0.802 +/- 0.116) was increased by 218.25% (P < 0.01) than that in normal skin (0.252 +/- 0.067). The expression of the gene in hypertrophic scar (0.628 +/- 0.144) was higher by 149.21% (while, P > 0.05) than that in normal skin. The expression of TGF-beta1 in keloid and hypertrophic scar were more than that in normal skin. The expression of fibrillin 1 was related to that of TGF-beta1 positively (r = 0.820, P < 0.01). Fibrillin 1 protein was stained positively in basic membranes, endothelial cells, fibroblasts and extracellular matrix of skin tissues. In dermal, the protein levels of fibrillin 1 in keloid (0.117 +/- 0.042) was decreased than those in normal skin (0.185 +/- 0.043) and hypertrophic scar (0.181 +/- 0.048), the inhibition rates were 36.76%, 35.36% respectively (both P < 0.01).
CONCLUSIONSThe expression of fibrillin 1 in keloid was changed and related to the expression of TGF-beta1 positively, which appears that fibrillin 1 was a cicatrix specific gene. Fibrillin 1 might play an important role in the formation of keloid.
Cicatrix, Hypertrophic ; metabolism ; pathology ; Fibrillin-1 ; Fibrillins ; Humans ; Keloid ; metabolism ; pathology ; Microfilament Proteins ; metabolism ; RNA, Messenger ; genetics ; Transforming Growth Factor beta1 ; genetics ; metabolism
7.A new protein Girdin in tumor metastasis.
Jing WANG ; Li FU ; Feng GU ; Yong-Jie MA
Chinese Medical Journal 2010;123(13):1786-1788
Cell Movement
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genetics
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physiology
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Humans
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Microfilament Proteins
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genetics
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metabolism
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Models, Biological
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Neoplasm Metastasis
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genetics
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physiopathology
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Protein Binding
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genetics
;
physiology
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Proto-Oncogene Proteins c-akt
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genetics
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metabolism
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Vesicular Transport Proteins
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genetics
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metabolism
8.Involvement of fascin-1-mediated autophagy in the biological behavioral of endometrial cells.
Xiaomei LUO ; Wei CHENG ; Shizhang WANG ; Zhihong CHEN
Journal of Central South University(Medical Sciences) 2018;43(9):957-963
To explore the mechanism for the role of autophagy in endometriosis, and to provide a theoretical basis for prevention and treatment of endometriosis.
Methods: The endometrial CRL-7566 cells were treated with ATG5 siRNA, autophagic activator rapamycin and autophagic inhibitor 3-MA, respectively. The cell proliferation and invasion were detected by clonal formation, cell growth curve and MTT assay. The clinical specimens of endometriosis were collected from 20 cases. The expression of autophagy marker LC3II and autophagy substrate protein P62 were detected.
Results: Rapamycin inhibited the proliferation and clonal formation of CRL-7566 cells, while autophagy inhibitor 3-MA and ATG5 siRNA showed opposite effect. Moreover, rapamycin inhibited filopodia growth in endometriosis, whereas overexpression of filopodia-relevant protein fascin-1 inhibited the decrease in invasiveness caused by rapamycin. In clinical samples, we also found a significant decrease of LC3II while an increase in P62 compared with the control group.
Conclusion: Autophagy inhibition may contribute to an increase in endometrial cell proliferation and invasiveness. Autophagy activation could be a potential strategy for endometriosis therapy.
Autophagy
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drug effects
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genetics
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Carrier Proteins
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genetics
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metabolism
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Cell Line
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Cell Proliferation
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drug effects
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Endometriosis
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physiopathology
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Endometrium
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cytology
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Female
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Gene Expression Regulation
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Humans
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Microfilament Proteins
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genetics
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metabolism
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Microtubule-Associated Proteins
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genetics
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RNA-Binding Proteins
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genetics
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Sirolimus
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pharmacology
9.Cloning and bioinformatic analysis of TAGLN2 cDNA of Bufo japonicus formosus.
Hui ZHUGE ; Jin-Qiang YUAN ; Shu-Fang ZHANG ; Xian-Yu YANG
Acta Pharmaceutica Sinica 2013;48(2):250-254
To study the bioactive polypeptides included in Bufo skin and its secretions the plasmid skin cDNA library of adult Japanese toad Bufo japonicus formosus was prepared. The pSD64TR has been used as the vector and the cloning sites are Xho I and EcoR I. To screen cDNAs encoding bioactive components, the plasmid cDNA library was transformed into E. coli DH5 competent cells, and positive colonies were screened by colony PCR (polymerase chain reaction). The suspension of a single colony in LB medium was used as the template, SP6 (the upstream primer of the plasmid cDNA library) and a primer with Xho I site and polyT were used as the primers. As the result, 465 positive colonies out of 1 344 were obtained and their plasmid were collected and sequenced. By homologous analysis, it was found that one of the cDNAs encoding a peptide with high homolog with transgelin-2, which was registered in GenBank (accession number: JX197456), and it was indicated as a partial cDNA sequence with a deletion at the 5' end. The transcript is 997 bp consisting of 31 bp 5', 618 bp 3' untranslated region (UTR) and an open reading frame (ORF) of 348 bp encoding a polypeptide of 115 amino acids. In the putative protein product, there is a calponin homology domain, two cysteine residues for a disulfide bond and three a-helix domains, and five potential phosphorylation sites. The homologous analysis indicates 90% similarity with Xenopus (Silurana) tropicalis and 89% with Xenopus laevis, and 71%-85% with other species.
Amino Acid Sequence
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Animals
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Base Sequence
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Bufonidae
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genetics
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metabolism
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Cloning, Molecular
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Gene Library
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Microfilament Proteins
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chemistry
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genetics
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metabolism
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Muscle Proteins
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chemistry
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genetics
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metabolism
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Open Reading Frames
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Phosphorylation
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Phylogeny
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Plasmids
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genetics
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Sequence Homology, Amino Acid
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Skin
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metabolism
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Xenopus
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genetics
10.Isolation of Tara protein and its gene cloning.
Jian-ping LAN ; Yi LUO ; Yuan-yuan ZHU ; Jie SUN ; Xiao-yu LAI ; Jing-yuan LI ; Jian YU ; Ji-min SHI ; Mao-fang LIN ; He HUANG
Journal of Zhejiang University. Medical sciences 2004;33(6):486-490
OBJECTIVETo isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene.
METHODSThe co-immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI-TOF mass spectrometry for protein identification. The candidate gene was amplified by temperature-gradient PCR from human testis cDNA library and then cloned into pEGFP-C2 vector for eukaryotic expression. The amplified gene was verified by direct sequencing and GFP-tagged protein was confirmed by immunoblotting.
RESULTSTara protein with the size of 68 kD was identified from the TRF1 precipitate. The candidate gene amplified from cDNA library was about 1.7 kb as expected. Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869). GFP-tagged fusion protein was about 100 kD. Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase.
CONCLUSIONTara might be an interacting protein with TRF1. However, further investigation would be required to confirm if they were bona fide partners.
Cloning, Molecular ; HeLa Cells ; Humans ; Microfilament Proteins ; genetics ; isolation & purification ; metabolism ; Protein Binding ; Telomeric Repeat Binding Protein 1 ; chemistry ; genetics ; metabolism