scholarly journals CLONING AND EXPRESSING HG-CSF (HUMAN GRANULOCYTE COLONY STIMULATING FACTOR) BEING FUSED WITH FERRITIN HEAVY-CHAIN IN E. COLI

2009 ◽  
Vol 12 (9) ◽  
pp. 47-53
Author(s):  
Hieu Thi Phuong Nguyen ◽  
Linh Thuy Lien ◽  
Thuoc Linh Tran
Keyword(s):  
Heavy Chain ◽  
Cloning And Expression ◽  
Form Expression ◽  
Gene Encoding ◽  
E Coli ◽  
Ferritin Heavy Chain ◽  
Tev Protease ◽  
Sds Page ◽  
Human Ferritin ◽  
Stimulating Factor

G-CSF is a cytokine that stimulates the proliferation, differentiation, function of mature neutrophils and is generally used for treatment of neutropenia in cancer patients under chemotherapy. In this study, we report results on the cloning and expression of hG-CSF being fused with the heavy-chain (FTN-H) of human ferritin, which had been showed to be capable of facilitate the folding of several human protein expressed in E. coli. The hg-csf gene and gene encoding FTN-H were inserted into plasmid pET28a to form expression vector PET-FHG. 6xHis tag and TEV sequence (being recognized and cleaved by TEV protease) was added between of G-CSF and FTN-H to facilitate G-CSF purification and recovery afterwards. PET-FHG was transformed into E. coli BL21(DE3). The expression of hG-CSF was induced by IPTG and confirmed by SDS-PAGE and Western blot using anti-hG-CSF antibody.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Construction of Prokaryotic Expression Vector Containing Flocculation Gene FLO5 and Expression of FLO5 in E. coli

2013 ◽  
Vol 310 ◽  
pp. 157-161 ◽  
Author(s):  
Jie Xue ◽  
Wen Qiang Wei ◽  
Dong Yan Zhang ◽  
Yong Li Li ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Prokaryotic Expression ◽  
Genomic Dna ◽  
Expression Vector ◽  
Gene Fragment ◽  
Form Expression ◽  
Protein Fragments ◽  
E Coli ◽  
Sds Page ◽  
Prokaryotic Expression Vector

FLO5 has been identified as a dominant flocculation gene. The goal of this study is to clone the FLO5 gene from Saccharomyces cerevisiae and express it in E. coli. In this study, the FLO5 gene amplified by PCR from S. cerevisiae was cloned into prokaryotic expression vector pET-28a to form expression vector pET28a-FLO5, finally, transferred into E.coli BL21. Methods: FLO5 gene was amplified by PCR from genomic DNA extracted from Saccharomyces cerevisiae. The amplified FLO5 gene fragment was then recombined with clone vector pMD18-T to form clone vector pMD18-T-FLO5 amplified in E.coli JM109. After confirmed with sequencing, FLO5 fragment cut out from pMD18-T-FLO5 by enzyme EcoRI and NotI was recombined into expression vector pET-28a to form vector pET28a-FLO5. Vector pET28a-FLO5 was then transferred into E. coli BL21 and protein FLO5 was expressed in E. coli BL21 by the induction with IPTG. Expressed protein fragments separated by SDS-PAGE showed a band with the size of protein FLO5 suggesting the expression of gene FLO5. with the expected This study will lay the foundation for further research in studying flocculating effect of exogenous protein expressed by genetic engineering and making new flocculating agent through recombinant engineering.

Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha

2017 ◽  
Vol 184 (4) ◽  
pp. 1286-1307 ◽  
Author(s):  
Valentina Ganeva ◽  
Bojidar Galutzov ◽  
Boyana Angelova ◽  
Manfred Suckow
Keyword(s):  
Heavy Chain ◽  
Hansenula Polymorpha ◽  
Ferritin Heavy Chain ◽  
Human Ferritin ◽  
Human Ferritin Heavy Chain

scholarly journals VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

2005 ◽  
Vol 11 (1) ◽  
pp. 61-66
Author(s):  
Ira Djajanegara ◽  
Wayan Artama ◽  
Retno Lestari ◽  
Sabar Pambudi
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Restriction Site ◽  
Standard Procedure ◽  
Pcr Amplification ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Coding ◽  
Sds Page ◽  
Encoding Protein

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization

Gene ◽  
1992 ◽  
Vol 111 (2) ◽  
pp. 255-260 ◽  
Author(s):  
M.A. Bevilacqua ◽  
M. Giordano ◽  
P. D'Agostino ◽  
C. Santoro ◽  
F. Cimino ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Functional Characterization ◽  
Ferritin Heavy Chain ◽  
Human Ferritin ◽  
Encoding Gene ◽  
Human Ferritin Heavy Chain

scholarly journals Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

2013 ◽  
Vol 16 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Trang Thi Phuong Phan ◽  
Anh Le Tuan Nguyen ◽  
Hoang Duc Nguyen
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Fusion Protein ◽  
Expression System ◽  
Pharmaceutical Products ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
E Coli ◽  
B Subunit ◽  
The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

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