Dash, Sagarika and S., Srimeenakshi (2017) Cloning and Co-Expression of hG-CSF and hSCF in E.coli. MTech thesis.
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Granulocyte Colony Stimulating Factor (G-CSF) is a hematopoietic growth factor that controls the proliferation, differentiation and the function of neutrophils. G-CSF is clinically used in human for the treatment of neutropenia in diseases such as AIDS, aplastic anaemia, myelodysplastic syndrome, and congenital or chemotherapy-induced neutropenia. However, the bioactivity and stability of commercially available recombinant hG-CSF such as Filgrastim and Lenograstim are lower than endogenous G-CSF. The objective of the present study is to express recombinant hG-CSF in E.coli along with SCF as a fusion partner to improve the bioactivity. In the present study, in silico analyses were performed for finding the possible mutant variants of G-CSF which may increase the stability of the recombinant hG-CSF for its incorporation into hG-CSF by site-directed mutagenesis and also analyse the binding affinity of the G-CSF-SCF fusion protein with G-CSF receptor (G-CSF-R) and SCF receptor (SCF-R). A total of 5 mutant variants of hG-CSF was generated and docked with GCSF-R using Hex dock 8.0 software. In order to analyse the binding affinity of the G-CSF-SCF fusion protein, docking analyses of the fusion protein with GCSF-R and SCF-R were performed using patch dock server. Human G-CSF gene (547 bp) was isolated from human Umbilical Cord Blood and U-87 cell line. The hG-CSF gene was cloned into TOPO-TA vector for sequencing followed by cloning into the pET14b vector for expression using Nde I and Bam HI restriction ends. Human GCSF gene was then end modified to fuse with the fusion partner. Besides, Human SCF gene (567 bp) was purchased from GenScript in the pUC57 cloning vector. It was restriction digested from the pUC57 vector using Bam HI and Xho I restriction enzymes. The restriction digested hG-CSF, SCF inserts were inserted into pET14b vector containing Nde I and Xho I restriction ends. The ligated expression vectors were then transformed into chemically competent DH5α cells followed by plasmid extraction and transformation into expression host E.coli BL21 (DE3).The expression of human G-CSF and G-CSF-SCF protein in E.coli was confirmed using SDS-PAGE analysis. Further expression profile of the proteins was optimized to increase the protein expression. From the in silico analysis, it was found that the mutant variant 5 may have improved biostability than the wild type G-CSF variant. The study was also found that the G-CSF-SCF fusion protein has high binding affinity to G-CSF-R and SCF-R from the global energy values. Furthermore, increased level of G-CSF and G-CSF-SCF fusion protein observed under optimized IPTG concentration of 1mM, post-induction duration of 8h and at 3% of ethanol concentration.
|Item Type:||Thesis (MTech)|
|Uncontrolled Keywords:||G-CSF; SCF; Fusion protein; In silico analysis; BL21DE3; cloning; transformation; expression; optimization;|
|Subjects:||Engineering and Technology > Biomedical Engineering|
|Divisions:||Engineering and Technology > Department of Biotechnology and Medical Engineering|
|Deposited By:||Mr. Kshirod Das|
|Deposited On:||23 Oct 2017 12:14|
|Last Modified:||23 Oct 2017 12:25|
|Supervisor(s):||Gupta, Mukesh Kumar|
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