Recombinant polypeptide XTEN as a suitable fusion partner human growth hormone


Please provide details of current and previous research experience, e.g. vacation scholarship, undergraduate project (no more than 300 words).

I Have Successfully completed a training program on "DNA Fingerprinting by Restriction Fragment Length Polymorphism (RFLP) and Short Tandem Repeats (STR)" organized by the "Department of Biotechnology", Manipal University, Dubai Campus, UAE. Also, while studying bioinformatics, I learnt how to access the data archives of genomes and proteins, the tools that have been developed to work with these archives, and the kind of questions that these data and tools can answer. I have emerged with a sense of optimism that the data and methods of bioinformatics can create profound advances in our understanding of life, and the improvements in health sciences. While at undergraduate level, I gained hands on experience in techniques underpinning rDNA technology such as preparation of E.coli competent cells and synthesis of cDNA using RT-PCR. I have also dealt with the practical fundamentals of in vitro culturing of animal and plant cells and covered types of culture, biology of cultured cells, procedures, methods and applications which made me familiar with sterile techniques and media preparation. I have had the opportunity to work with HeLa cell lines which involved their cryopreservation, subculturing, revival and quantitation using a hemocytometer. All of this enabled me to learn about the principles of plant and animal cell culture, commercial applications and various methods to generate the same. I have even completed a mini-project, working in pairs, to sub-clone, mutate, express and purify the Green Fluorescent Protein, thereby gaining practical competence in techniques such as plasmid DNA preparation and quantification; restriction enzyme digestion; site-directed mutagenesis; PCR; DNA sequencing; Agarose Gel Electrophoresis; SDS-PAGE and Western Blotting; protein purification by Affinity Chromatography. Hence, I feel that I have gained a substantial amount of practical laboratory experience that would qualify me as a potential PhD applicant to work with your renowned research team.

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Outline the reasons why you wish to study for a PhD and the career you intend to pursue (no more than 500 words).

I have always wanted to achieve something significant. I am ambitious and I yearn to provoke myself to achieve complex goals. I have always loved to discover and learn about new findings. I have never lost my naive interest in scientific discoveries taking place around the world and so I believe I that would become a successful researcher. I possess an unceasing initiative within myself that propels me to explore new things and learn more about them, and I love research, and hence feel that a doctorate will be perfect for me. I also wish to improve myself and my life. I want to enhance my abilities to comprehend and solve real life problems, increase my confidence and communication skills and develop competence that may lead me to a better job. I think a PhD just fits me. Some people are committed to pursue a doctorate and I feel I am one of them. I have always worked on numerous mini research projects as hobbies, either in the form of investigating plants or finding the reasons behind various universal phenomena. I have a characteristic quest for knowledge and an ardent penchant for reading various books on specific topics. In fact, I have had an everlasting fascination about Biology.

The objective on completion of my PhD is to pursue a very successful and a proficient career, that would allow me to be recognized as an individual who positively influenced the lives of all the people and as well as organizations that I was associated with. I intend to accomplish all tasks with a very positive attitude, a lot of devotion, as well as an ardour to make a difference. I want to be exposed to a wide range of research areas. In the initial period of my career, I would like to gain vast knowledge and an ample amount of practical experience. My ultimate goal would be to contribute to cutting-edge research in the Life Sciences and to develop novel drugs and therapeutic compounds for the benefit of mankind. I want to pursue a satisfying career in the field of biological sciences, which is aiding in the development of extensive innovations in the medical, industrial and agricultural sectors that were earlier extremely time-consuming and labour-intensive. Ideally, I would enter into a stage in my professional career where I would be acknowledged as a well renowned research scientist. I would gain immense contentment and satisfaction by learning that my contribution to research has had a beneficial impact on the existence of all those affected. If I succeed in achieving all of my goals completely, I should be able to acquire an abundance of veneration towards the latter part of my life. I hope to lead a long life to be able to quench my thirst for knowledge and persevere in my quest for a fruitful career in Biotechnology, thus giving back to the society, and leaving a lasting impression on it for ages to come.

Word Count: 493



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What is your research question? (no more than 100 words)

It is highly desirable to express high levels of recombinant human growth hormone (somatropin) in bacteria such as Escherichia coli, owing to its therapeutic potential. However, the expressed protein often tends to aggregate within the bacteria and form inclusion bodies. This phenomenon impedes the isolation and purification of the functional recombinant protein, hence limiting its availability for medical applications. Attachment of an XTEN sequence as a fusion partner to the target protein sequence could lead to the production of soluble and biologically active somatropin, thus reducing the number of laborious and time-consuming steps employed in its purification and refolding procedures.

Word Count: 100


Why do you think this area of research is interesting? (no more than 250 words)

Human Growth Hormone (hGH) is a 191 amino acid single chain polypeptide of molecular weight 2,200Da, produced by the anterior pituitary gland. It is used in the treatment of clinical conditions such as pituitary dwarfism in children lacking sufficient endogenous levels of hGH to correct short stature. Scientific evidence has also facilitated approval of its administration for the treatment of pathological conditions such as chronic renal insufficiency, osteoporosis and severe burns. Lately, it has also been demonstrated to be effective in the treatment of HIV related infection.

Such potential applications of the hormone have generated widespread clinical and industrial interest, and hence, its demand as a therapeutic protein has risen exponentially.

The advent of rDNA technology has enabled industrial production of the hormone by expressing it in E.coli and this helped to partly overcome its limited supply for clinical applications, as earlier the only reliable source of the protein was the human pituitary gland(Gary and Headon, 1994).

However, several problems associated with the expression of the protein in E.coli still exist. One such major problem is its expression in an insoluble form in inclusion bodies which makes its recovery as a functional protein significantly difficult.

Application of a novel experimental strategy, such as XTEN technology, to enhance the solubility of the expressed recombinant protein and reduce the fraction of inclusion bodies would afford cost-effective and efficient recovery and purification of the therapeutic protein, thus undoubtedly simplifying and maximising its large-scale production in a biologically active form, which would also promote further experimental studies.

Word Count: 250


How do you propose to address this research question? (no more than 750 words)

Fusion of an XTEN sequence to a therapeutic peptide exenatide has been shown to enhance the solubility and yield of the attached protein partner, when expressed in E.coli . A similar approach could be applied to somatropin, by employing the experimental strategy outlined below:

1). Preparation of DNA fragments encoding somatropin and XTEN -

DNA sequence of somatropin would be prepared by using the Phosphoramidite method of Chemical DNA Synthesis , based on its known amino acid sequence.

The gene encoding the 864aa XTEN fragment would be synthesised as described in the literature . The XTEN and somatropin DNA fragments generated would then be sequenced using the dideoxy chain-termination method to ensure their correctness.

2). Fusion of somatropin and XTEN followed by ligation into a suitable vector -

The DNA sequence for somatropin would be fused to a preceding DNA sequence encoding for a Tobacco Etch Virus(TEV) restriction site, using adaptors/linkers or overlap PCR. Following this, the resulting TEV/somatropin sequence would be fused to another preceding DNA sequence encoding for the C.thermocellum Cellulose Binding Domain(CBD), again using overlap PCR.

The CBD-TEV/somatropin sequence thus generated would be ligated into a pET30 derived XTEN expression vector(eg.pCW0359) by addition of appropriate restriction sites,(eg. Bsa1 and Bba1) to the vector and the fusion sequence, ensuing in the generation of a CBD-TEV/somatropin-XTEN fusion sequence. The resulting constructs would then be subjected to restriction enzyme digestion for analysis and verification.

3). Transformation of appropriate bacterial cells followed by screening and selection of positive clones -

The recombinant expression vector would be transformed into E.coli DH5α cells and subjected to DNA sequencing to ensure fidelity, followed by transformation into competent protein expression host cells. Eg. E.coli Gold BL21(DE3). Alternatively, E.coli cells could be made competent by electroporation or CaCl2 transformation.

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The cell culture would then be plated onto LB-Kanamycin agar plates, which would allow growth of only positively transformed cells as bacterial colonies. This would be possible as the kanamycin resistance gene in the expression vector would confer antibiotic resistance to the transformed cells.

Positive clones would then be selected for verification by colony PCR and agarose gel electrophoresis.

4). Induction of protein expression in the positively transformed host cells -

The effectively transformed host cells would then be cultured in a kanamycin containing nutrient media placed in a shaking incubator to further ensure growth of only transformed cells.

The growth of the culture could be determined by measuring its optical density(O.D) at 600nm. At this wavelength, 1 O.D would represent 0.8Ã-109 cells/ml.

The cells would then be induced by IPTG to over express the fusion protein. This would be achieved by binding of IPTG to the T7 repressor protein or its displacement from the operator region of the bacterial DNA.

5). Recovery of transformed cells and isolation of the fusion protein -

Following induction, the cells would be harvested by centrifugation and then lysed to release the fusion protein of interest along with other intracellular components.

The cells could either be ruptured mechanically(homogenisation, micro-fluidisation, sonication) or lysed chemically(lysosyme + EDTA + SDS).

Removal of cell debris would be accomplished by differential centrifugation, which would yield a clear lysate containing the soluble protein, as an expected consequence of the unique characteristics conferred by the XTEN sequence for maximum product recovery.

The lysate would then be heated, followed by addition of TEV protease to the sample, in order to cleave the CBD and TEV sequences from the fusion protein.

6). Purification, concentration and verification of the recombinant protein -

It would be prudent to employ a minimum of three chromatography steps to obtain a virtually pure therapeutic protein meant for parenteral administration, as the presence of even trace amounts of contaminants could lead to adverse clinical reactions.

The protein sample would be applied onto ion-exchange and hydrophobic interaction chromatography columns to purify the target protein on the basis of its net charge and hydrophobic characteristics respectively. The target protein could then be concentrated by centrifugation and/or filtration.

The fidelity of the amino acid sequence of the fusion protein would be determined by N-terminal sequencing followed by non-reducing SDS-PAGE analysis to preserve its native structure and estimate its molecular weight.

7). Determination of physical properties possessed by the fusion product -

Mass Spectroscopy and Circular Dichroism would be employed to determine precise mass of the protein and presence/absence of any secondary structural features respectively.

Gel Filtration Chromatography coupled with Multi-angle Light Scattering would then be used to determine absolute molecular mass of the fusion protein.

This strategy could potentially ease and expedite downstream processing of the therapeutic recombinant protein.

Word Count: 750


What scientific considerations led you to choose the laboratory and supervisor for your research project? (no more than 150 words)

There are indeed not one but various scientific reasons that led me to choose this 18,000 sqft research area and such a pioneer as you for my research project. The fact that your research is focused towards the discovery and development of therapeutic proteins with extended in vivo half-lives has generated a potential interest in me to further exploit XTEN technology to facilitate effective protein expression and recovery. I would be privileged to work in your state-of-the-art laboratory buildings which offer modern, flexible and fully serviced space that perfectly fits the need of biosciences research. I would consider myself honoured to join your mission of creating and advancing research and development that is expected to benefit millions of those suffering worldwide. I believe that your company has all the facilities and services needed to increase productivity and efficiency, stimulate and enable innovation and facilitate commercialisation of life saving biotherapeutics.