Cloning and Co-Expression of hG-CSF and hSCF in E.coli Sagarika Dash and Srimeenakshi S Dept. of Biotechnology and Medical Engineering National Institute of Technology Rourkela Cloning and Co-Expression of hG-CSF and hSCF in E.coli Dissertation submitted in partial fulfilment of the requirements of the degree of Master of Technology in Biotechnology by Sagarika Dash (Roll Number: 215BM2007) & Srimeenakshi S (Roll Number: 215BM2011) based on research carried out under the supervision of Prof. Mukesh Kumar Gupta May, 2017 Department of Biotechnology and Medical Engineering National Institute of Technology Rourkela Dept. of Biotechnology and Medical Engineering National Institute of Technology Rourkela Dr. Mukesh Kumar Gupta Associate Professor May 30, 2017 Supervisor’s Certificate This is to certify that the work presented in the dissertation entitled “Cloning and Co- Expression of hG-CSF and hSCF in E.coli” submitted by “Sagarika Dash”, Roll Number: 215BM2007 and “Srimeenakshi S”, Roll Number: 215BM2011 , is a record of original work carried out by her under my guidance and supervision in the partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at the Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela. Neither this dissertation nor any part of it has been submitted earlier for any degree or diploma to any institute or university in India or abroad. Mukesh Kumar Gupta i Declaration of Originality We, Sagarika Dash, Roll Number 215BM2007 and Srimeenakshi S, Roll Number 215BM2011 hereby declare that this dissertation entitled Cloning and Co-Expression of hG-CSF and hSCF in E.coli presents our original work carried out as a master students of NIT Rourkela and, to the best of my knowledge, contains no material previously published or written by another person, nor any material presented by us for the award of any degree or diploma of NIT Rourkela or any other institution. Any contribution made to this research by others, with whom we have worked at NIT Rourkela or elsewhere, is explicitly acknowledged in the dissertation. Works of other authors cited in this dissertation have been duly acknowledged under the sections “Reference” or “Bibliography”. We have also submitted our original research records to the scrutiny committee for evaluation of our dissertation. We are fully aware that in case of any non-compliance detected in future, the Senate of NIT Rourkela may withdraw the degree awarded to me on the basis of the present dissertation. May 30, 2017 Sagarika Dash NIT Rourkela Srimeenakshi S ii Acknowledgement Thinking and Feeling appreciation signifies that we believe in someone’s existence rather than the divine who rules everything. This thesis entitled “Cloning and Co-Expression of hG-CSF and hSCF in E.coli” is the successful outcome of guidance and support bestowed on us throughout our work. We would like to express our most sincere gratitude to every single being those devoted their time for successful completion of our thesis. We would like to show my foremost gratitude towards my Guide Prof. Mukesh Kumar Gupta who helped me with my thesis in every possible stage and encouraged me to do better. We would also like to thank Ph.D. scholar Bijayalakmi Sahoo, and Senior Research Fellow Dr. Rakesh Bhaskar and all our lab mates for their painstaking effort for the completion the thesis. Finally, we would not miss to thank heartily to our parents and close friends for their unconditional love and encouragement shown towards me and completion of the thesis. Last but not the least, we would like to thank the Department of Biotechnology and Medical Engineering for providing such a stimulating atmosphere and nice work environment. May 30, 2017 Sagarika Dash NIT, Rourkela Roll Number: 215BM2007 Srimeenakshi S Roll Number: 215BM2011 iii Abstract 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 optimised 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 iv the global energy values. Furthermore, increased level of G-CSF and G-CSF-SCF fusion protein observed under optimised IPTG concentration of 1mM, post-induction duration of 8h and at 3% of ethanol concentration. Key words: G-CSF, SCF, Fusion protein, In silico analysis, BL21DE3, cloning, transformation, expression, optimization. v Contents Supervisor’s Certificate ........................................................................................................ i Declaration of Originality ................................................................................................... ii Acknowledgement .............................................................................................................. iii Abstract ............................................................................................................................... iv List of Figures ................................................................................................................... viii List of Tables ....................................................................................................................... x Chapter 1 .............................................................................................................................. 1 Introduction.......................................................................................................................... 1 Chapter 2 .............................................................................................................................. 6 Literature Review ................................................................................................................ 6 2.1 Human granulocyte colony stimulating factor .............................................................. 6 2.1.1 The structure of G-CSF .............................................................................................. 6 2.1.2 The genetics of G-CSF ............................................................................................... 8 2.1.3 The functions of G-CSF ............................................................................................. 8 2.1.4 Clinical Importance of G-CSF .................................................................................... 8 2.2 Hematopoietic growth factors........................................................................................ 9 2.3 G-CSF fusion protein ................................................................................................... 10 2.4 Stem Cell Factor (SCF) ............................................................................................... 10 2.4.1 Biology of Stem Cell Factor ..................................................................................... 11 2.5 Synergistic effect of G-CSF and SCF .......................................................................... 11 Chapter 3 ............................................................................................................................ 15 Objectives .........................................................................................................................