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STATEMENT OF PURPOSE
When we boil an egg, the proteins in it fold resulting in a solid mass, but never go back to its original liquid state after cooling. Similarly, Biochemists have always cursed the lumps of proteinaceous mass left over in tubes. Yeasts are used in leavening of bread and fungi turn the bread stale. These facts fascinated me very early in life. As a teenager at school, biology fascinated me and I visualized a career in research. I opted for biology after my school and later joined University for Bachelors in Science with Biotechnology, Biochemistry, Chemistry. The sharing of ideas and new findings has always been a vital part of my undergraduate life. I opted for MSc in UK and what followed is learn and perform phase in my life. The course modules and thesis research project at Abertay University helped me in getting accustomed to latest technologies and enhanced my practical and theoretical knowledge.
M.Sc Project: Expression of Human Thioredoxin in K562 Erythroleukemic Cell Line
I have undertaken a 5 month research project for my MSc qualification. The project consisted of expression of thioredoxin in K562 cells. The human K562 erythroleukemia cell line is a multipotent haematopoietic precursor cell line derived from Chronic Myeloid Leukaemia (CML) patient in blast crisis. They K562 cells lack immunoglobulins, actively proliferate and are non adherent. The cell line taken as an experimental tool was routinely subcultured and the active form of protein was extracted using protease inhibitors. The extracted protein was subjected to separation using SDS-PAGE with a 12KDa marker. A western blot was performed to extract the thioredoxin protein. It was characterised by Immunodetection. The isolation of thioredoxin gene was initiated with RNA extraction and obtaining a cDNA by RT-PCR. Specific primers were designed and a PCR was performed to get the DNA. The thioredoxin gene amplified was to be confirmed with restriction enzymes BfuA1 and Mme1. The restriction digests were analysed on an agarose gel. The results of BfuA1 were accurate but the Mme1 enzyme did not cut the PCR product at the desired position. I figured out through literature reviews that The Mme1 enzyme blocks cleavage by CpG methylation when a sequence of TCCAACG nucleotides is encountered. This sequence was observed in the open reading frame of thioredoxin sequence at position 148 - 153 base pairs.
Also, the enzyme Mme1 usually cuts at 20/18 base pairs downstream of the usual cut site (173bp). This explained the inefficient working of Mme1 restriction enzyme. The verified gene obtained was ligated into a plasmid and later cloned in JM109 chemically competent E.coli. The colonies obtained were analysed for the positive clones. These clones were later analysed by arabinose induction for the thioredoxin protein. The amassing expression of thioredoxin protein in the K562 cells may pave the way for the introduction of the several therapeutic applications. This project work helped me in developing my abilities of prioritizing tasks, troubleshooting experiments when required and performing literature reviews to strengthen my theoretical knowledge.
The Bioinformatics and Functional Genomics course modules helped me gain knowledge relating to protein structure and function. The several course works undertaken included -
- Protein Identification and analyses by Peptide Mass Fingerprinting and MALDI-TOF. A detailed coursework on the procedures and parameters involving database searching using MS-Fit and Mascot.
- Analyzing the patterns and structural variations in different protein structures using Swiss PDB viewer (DeepView) and RCSB protein data bank.
- From sequence to structure and function - a coursework involving random protein sequence which is analysed for for its structure and function using the databases and EBI tools like ExPASy, SwissProt, UniProt, Ensembl, etc
- Chaperone Mediated Protein Folding - A case review on molecular chaperones involved in invivo protein folding, describing the classes of heat shock proteins, calnexin and calreticulin.
At this juncture, I am interested in taking forward this streak of interest and exposure in protein biology to a level further. I would like to work under your supervision on the project - ‘Denovo design of novel Repeat Proteins'. I would like to learn and understand the protein binding abilities of Leucine Rich Repeat (LRR) proteins. I would be eager to utilize my technical, dry lab and bioinformatic skills coupled with literature reviews to understand the protein folding involved. I am interested in learning and acquiring new computational skills by using different tools and databases which will help in designing novel LRR proteins that may serve as scaffolds for the development of novel protein binders.. I would be eager to dedicate my innate interest and use my potential in making outcomes and research seem that way. My aim would be a balanced computational and experimental output of the project, with your supervision to achieve a successful outcome in the years to come.
I hope to turn this interest into a learning experience when I start the post graduate research at the Division of Biochemistry, Department of Chemistry, Lund University, Sweden. I want to put to practice my abilities and also learn from each and every person young/old who comes across in my lab/research. I want to be a sincere, ever enthusiastic, hardworking and an achieving student in the coming years of my life to gain academic excellence and establish a career in research. Someone rightly said - “Scientific research has its origins in a fundamental human character - curiosity. It is essential though, to ask the right question”. Research in Protein Biology may raise many questions and I would like to be one working toward the answers. I am eager and enthusiastic to start the project on a sooner note and am readily available as of now. I would be pleased if am accepted for this project and can revive my interests in protein Biology.