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This document is a thesis submitted by Stuart A. Deane to Trinity College Dublin in partial fulfillment of the requirements for a Masters in Bioengineering. The thesis describes the development of a bioengineered, decellularised xenograft for mitral valve replacement. Through a literature review, additional constructs were designed to standardize the surgical procedure and establish natural geometry. A decellularization protocol was assessed for its effects on mechanical properties and microstructure of porcine mitral valves. Uniaxial tensile testing showed the decellularization process did not significantly change the mechanical properties of the leaflets or chordae. Histological staining also showed no visible changes to microstructure or remaining nuclear material after decellular
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This document discusses momentum and its relationship to mass and velocity. It defines momentum as being equal to mass multiplied by velocity, and explains that momentum is a vector quantity. It also discusses impulse, which is defined as the change in momentum, and explains how impulse is related to force and time through the equation: Impulse = Force x Time. The document notes that momentum is always conserved during interactions and collisions.
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From the Stem Cell workshop at the Bio-Link Summer Fellows Forum 2011
presenters: Edie Kaeuper, Carin Zimmerman, Golnar Afshar, Samantha Croft, & Bill Woodruff
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Warren Campbell is seeking MD/PhD degrees to integrate clinical and research careers in cancer biology, stem cell biology, neurobiology, or biochemical engineering. He has a strong academic record including honors degrees from Gettysburg College and study abroad. Campbell has extensive research experience in cancer and neurodegenerative diseases at the National Institutes of Health and as an undergraduate. He also has teaching experience as a laboratory assistant and peer mentor in biology and chemistry courses.
Siddarth Kaul is being strongly recommended for a full time position by his professor Anuj Chauhan. As a teaching assistant and research assistant, Siddarth has demonstrated excellence through high quality novel research and academic achievement with a GPA of 3.94. As a graduate student, Siddarth is in the top 5% and impressed with his undergraduate credentials from Panjab University in India. In Professor Chauhan's lab, Siddarth developed a novel solution to remove toxic preservatives from eye drops using particles placed in the bottle dropper, which has potential for immense impact on hundreds of millions of patients. Siddarth is an intelligent, creative, and hard working student with strong research and communication
Jude Tunyi is a third-year biochemistry, chemistry, and cellular and molecular biology student at the University of Washington seeking an internship. He has research experience in developing computational models to increase insulin stability using molecular dynamics simulations. He also has experience developing bacterial vaccines and tutoring chemistry. His skills include Linux, coding languages like Python and Matlab, and molecular dynamics software like GROMACS and AMBER. He has presented his research widely at conferences.
V. Hemambujavalli is seeking an opportunity in an organization where she can utilize her skills in microbiology. She has a M.Sc. in Microbiology from SASTRA University and 3.5 years of experience as a research analyst at Thomson Reuters. Her skills include laboratory experience in various areas of microbiology as well as strong communication, leadership, and analytical abilities.
This document provides information on a dissertation submitted in partial fulfillment of a BBA degree at Heriot-Watt University in Dubai. The dissertation examines the levels and quality of environmental and social disclosure by UAE companies. It includes chapters on the introduction and background, literature review, research methodology, data interpretation and results, and conclusion. The dissertation uses qualitative content analysis to analyze annual reports from 20 UAE companies to assess their economic, environmental, and social disclosures. The interpretations find that while social disclosures are often lacking, the selected companies provide detailed information that could be useful for future research. The summary provides a high-level overview of the dissertation structure and research topic.
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Để xem full tài liệu Xin vui long liên hệ page để được hỗ trợ
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M. Govardhanan is seeking a challenging career in biotechnology where he can utilize his 8+ years of experience in mammalian cell culture. He has worked at Syngene International, Lupin Limited, and Biocon Biopharmaceuticals in roles involving cell culture, downstream processing, batch execution, and equipment qualification. He has hands-on experience with bioreactors up to 2KL scale and single-use bioreactors up to 500L scale. M. Govardhanan holds an MSc in Biotechnology and a BSc in Biotechnology.
Assessing the Impact of a Virtual Lab in an Allied Health Program.pdfElizabeth Williams
The document is a thesis that assesses the impact of a virtual lab in an allied health program. It conducted a mixed-methods study collecting quantitative and qualitative data from students who used an online virtual lab to prepare for hands-on microbiology labs. The data showed students had a positive attitude towards the virtual lab and found it helped prepare them, make skills acquisition easier and faster, and would use it again without needing incentives. The virtual lab allowed students to visualize procedures and reactions outside of the traditional lab setting, which enhanced their preparedness and performance in the hands-on lab.
Writing Third Year Project Report: Some Handy GuidelinesMithil Fal Desai
The document provides guidelines for writing a third year project report. It discusses the different types of research projects including fundamental, descriptive, empirical, quantitative, applied, analytical and qualitative. It outlines the typical sections of a project report such as the title page, declaration, certificate, acknowledgment, index, abstract, introduction, experimental, results and discussion, conclusion and references. For each section, it provides details on the content and formatting. It emphasizes writing in a clear, concise and organized manner with proper citations and references. The document aims to help students structure their project reports and effectively communicate their research.
Warren Campbell is seeking MD/PhD degrees to integrate clinical and research careers in cancer biology, stem cell biology, neurobiology, or biochemical engineering. He has a strong academic record including honors degrees from Gettysburg College and study abroad. Campbell has extensive research experience in cancer and neurodegenerative diseases at the National Institutes of Health and as an undergraduate. He also has teaching experience as a laboratory assistant and peer mentor in biology and chemistry courses.
Siddarth Kaul is being strongly recommended for a full time position by his professor Anuj Chauhan. As a teaching assistant and research assistant, Siddarth has demonstrated excellence through high quality novel research and academic achievement with a GPA of 3.94. As a graduate student, Siddarth is in the top 5% and impressed with his undergraduate credentials from Panjab University in India. In Professor Chauhan's lab, Siddarth developed a novel solution to remove toxic preservatives from eye drops using particles placed in the bottle dropper, which has potential for immense impact on hundreds of millions of patients. Siddarth is an intelligent, creative, and hard working student with strong research and communication
Jude Tunyi is a third-year biochemistry, chemistry, and cellular and molecular biology student at the University of Washington seeking an internship. He has research experience in developing computational models to increase insulin stability using molecular dynamics simulations. He also has experience developing bacterial vaccines and tutoring chemistry. His skills include Linux, coding languages like Python and Matlab, and molecular dynamics software like GROMACS and AMBER. He has presented his research widely at conferences.
V. Hemambujavalli is seeking an opportunity in an organization where she can utilize her skills in microbiology. She has a M.Sc. in Microbiology from SASTRA University and 3.5 years of experience as a research analyst at Thomson Reuters. Her skills include laboratory experience in various areas of microbiology as well as strong communication, leadership, and analytical abilities.
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This document summarizes the experience and qualifications of Steven Brown, including over 7 years of experience in petrochemical and biopharmaceutical industries as a research scientist and project manager. He has strong technical skills in fermentation science, experimental design, automation, and analytical techniques. Brown is currently pursuing his PhD researching engineering carbon metabolism in yeast. He aims to develop an industrial yeast that can increase the product portfolio of cellulosic ethanol plants.
2015 cover letter + resume for environment jobsKyle Burwitz
The document is a cover letter from Kyle Burwitz applying for a job with a biology degree. He has a passion for science from a young age when observing ants. His broad education includes courses in conservation biology, neuroscience, and aquaculture. He enjoys group projects but can also work independently. Kyle is on the dean's list and his GPA has improved tremendously. He is excited to start his career and find a job that allows him and the company to succeed.
Development and validation of a predictive model for oncology hospital at-homeTÀI LIỆU NGÀNH MAY
Để xem full tài liệu Xin vui long liên hệ page để được hỗ trợ
: https://www.facebook.com/thuvienluanvan01
HOẶC
https://www.facebook.com/garmentspace/
https://www.facebook.com/thuvienluanvan01
https://www.facebook.com/thuvienluanvan01
tai lieu tong hop, thu vien luan van, luan van tong hop, do an chuyen nganh
This document provides information about a practical microbiology module for nursing students at GASHA private technical institute. The 4-hour per week module will cover basic microbiology principles including bacterial structures, growth, and identification. Students will learn laboratory techniques like staining, culturing, and antimicrobial testing. The module aims to help students recognize microbes that cause disease and apply diagnostic methods. Assessments include exams, quizzes, assignments, presentations, and lab reports. The course schedule outlines weekly practical lab activities focusing on topics like sterilization, media preparation, gram staining, and capsule staining.
Rachel Buckley is seeking a job in the biomedical field. She has experience as a teaching assistant and undergraduate researcher at Auburn University, where she is pursuing a Bachelor's degree in Biomedical Sciences. Her research involves using CRISPR/Cas9 to edit genome fragments in Drosophila melanogaster. She has also worked as a production assistant and sales associate. Buckley maintains a 3.48 GPA and is involved in various honors programs, research, and volunteer activities related to her field of study.
Kyle Pollard seeks a position in biological research, biotechnology, clinical studies or biology laboratory. He has a Bachelor's degree in Molecular and Cellular Biology from the University of Northern Colorado. His relevant skills include maintaining cell cultures, PCR, electrophoresis, immunohistochemistry, and microscopy. He has research experience in breast cancer biology and stem cell biology laboratories, where he performed techniques such as isolating RNA/DNA, PCR, and immunofluorescence microscopy. Kyle also has experience coordinating events and public speaking.
Vijay S. Dhawale is seeking a challenging career in IVF or microbiology. He has a M.Sc. in microbiology from Govt. Institute of Science in Aurangabad. He has over 2 years of experience as a junior embryologist and IVF technician. He is currently working as a junior embryologist at Aditya Birla Memorial Hospital in Pune. He has skills in IVF procedures, embryo culture, ICSI, and microbiological analysis techniques.
Wenjie Luo has extensive work and research experience developing biomedical engineering assays and experiments. She has worked at Suneris developing their hemostatic product VETIGELTM and conducted research at Columbia University and Cornell University on topics including tissue engineering, cancer treatment, vaccine development, and epigenetics. She received her M.S. in Biomedical Engineering from Columbia University and B.S. in Biological Engineering and minor in Biomedical Engineering from Cornell University.
InKForge Launch Event - 17 January 2012 - The University of Sheffield
Thesis_Final_Draft 6-24_with_DW_FINAL
1. Application of a Self-Cleaving Intein for Purification of Antimicrobial Peptides
through a Novel Affinity Tag
A Thesis Presented in Accordance with the Requirements for Graduating with Research
Distinction in Chemical and Biomolecular Engineering
By
Robert Wayne Gammon Pitman
Undergraduate Chemical and Biomolecular Engineering
The Ohio State University
2013
Oral Examination Committee
Dr. David W Wood,” Advisor” and Dr. Peter F Rogers
3. iii
Abstract
Increasing antibiotic resistance of bacteria presents an immediate global health
challenge, and has prompted the search for new therapeutics. Antimicrobial peptides,
small proteins with less than 100 amino acid residues, are one promising new class of
candidate molecules for antimicrobials. One obstacle to their study is lack of a reliable
method of recombinant purification in Escherichia coli, E coli. A higher yield and purity
of the peptide are necessary for E. coli efficiency in future scale-up. Previous work in the
laboratory of Dr. David Wood has developed a robust system for the purification of
recombinant proteins in E. coli based on self-cleaving purification tag technology. The
intein, which is a self-splicing protein segments, is engineered to become a viable
purification method.
In this study, a fusion protein consisting of a self-cleaving intein, protein of
interest and an affinity tag, choline binding domain (ClBD) is purified via a purification
column Q-sepharose. The ClBD specifically binds to Q-sepharose, by a bio-specific site
called the ligand binding domain (LBD). The purpose of this study is to possibly increase
the recombinant yield of active antimicrobial peptide by using a smaller affinity tag. This
system is an intriguing possibility for the purification of antimicrobial peptides because it
utilizes commercially available and FDA approved chromatography resins. One peptide,
Equine Defensin α-1 or DEFA1, was cloned into the pET-ClBD-I vector and expressed in
E coli BLR cells. Protein purification has been attempted and is ongoing via a Q-
4. iv
Sepharose ion-exchange column, which in this case acts as an affinity column. Once
purification is successfully performed the activity of the peptides will be tested using
broth microdilution and radial diffusion tests.
5. v
Dedication
This document is dedicated to Allyson Campbell my family. Without your
guidance and influence, I would not be where I am today.
This document would not be possible without any of the individuals whom gave
me a chance. Dr. Wood and those unmentioned, I cannot thank you all enough for your
support, assistance, and understanding. .
6. vi
Acknowledgements
I can't thank Dr. Wood enough for giving me the opportunity to do research under
his guidance. In my opinion he is a model professor who responds promptly to a student's
questions while providing honest, realistic feedback and surrounds himself with bright
students. I must profusely thank all the graduate students in Dr. Wood's lab and Dr.
Richard Lease because without all of your advice and guidance my research would not
have progressed with significantly slower results. Out of all my research experience, I
would conclude that the given opportunity to research under Dr. Wood was one of my
favorite experiences so far. The comradely yet distinct professionalism, where everyone
was willing to assist and advise me is a valuable interaction. One I have missed since
concluding my ROTC career. I would also like to thank all my Professors throughout my
Chemical Engineering academic career for being flexible and approachable. Dr. Scaccia,
I am not sure what you saw in me nearly a year ago but I can't thank you enough for
giving me a chance to work with you and share your advice. Your guidance has helped
me set up rally points and steps to get me from where I am not to where I want to be. Our
relationship gave me the resolve to attempt befriending all my professors. Without this
initiative, I would never have met Dr. Rogers who provided some of the best career
advice and support I could ask for.
To my friends and family, I thank you for always supporting my efforts
throughout this study, which often required unscheduled, extended evenings in the lab.
7. vii
Vitae
Work and Project Experience
Teaching Assistant for Freshman Engineering nanotechnology (Jan. 2013 –
Present) and Unit Operations lab (Apr. 2012- July 2012 & Jan. 2013 – Present)
Advising and/or teaching Proctor & Gamble’s Senior Design group (Spring term
2012 & Fall term 2012)
Senior design project for Givaudan to develop and build processes for a specific
use followed by presenting the specific processes to Givaudan (Jan 2012 – May
2012)
Research Experience
Dr. David W. Wood: Research thesis: Recombinant Expression of Antimicrobial
in E. coli (Summer 2012 - Present)
Dr. Palmer: Filtration of hemoglobin and the effects of cross-linking (Fall 2012)
Dr. Hadad: Synthesizing various molecules for nerve gas antidote and tracking
(Summer 2012)
Dr. Koelling: Biodegradable polymers and rheological properties (2011 - 2012)
Academic Recognitions
Fall 2012 .................................................................................Research Scholarship
Fall 2011 .................................................................................................Dean’s List
Fall 2010 .....................................Air Force R.O.T.C. Academic Excellence Award
June 2008 .......................................................................... Lakota East High School
Fields of Study
Major Field: Chemical and Biomolecular Engineering
8. viii
Table of Contents
Abstract..............................................................................................................................iii
Dedication........................................................................................................................... v
Acknowledgements............................................................................................................ vi
Vitae..................................................................................................................................vii
Table of Contents.............................................................................................................viii
1.0 Introduction................................................................................................................... 1
1.1 Proteins ................................................................................................................... 1
1.2 Inteins...................................................................................................................... 2
1.3 Antimicrobial Peptides and Purification................................................................. 4
1.4 Fusion Protein and Affinity Chromatography ........................................................ 6
2. Materials and Methods.................................................................................................. 11
2.1 Cloning ClBD-Intein-DEFA1 Vector................................................................... 11
2.2Expression of Protein............................................................................................. 14
2.3 Protein Purification of DEFA1 ............................................................................. 15
2.4 Defensin Activity via Radial Diffusion Assay...................................................... 17
3. Results and Discussion ................................................................................................. 19
3.1 Creation of pET-ClBD-I-DEFA1 ......................................................................... 19
3.2 Protein Purification............................................................................................... 21
4. Conclusions................................................................................................................... 23
Work Cited........................................................................................................................ 25
9. ix
List of Figures
Figure 1. Intein Function Upstream (left)........................................................................... 2
Figure 2.: C-terminal Cleaving ........................................................................................... 3
Figure 3: Host Cell (E. coli)............................................................................................... 5
Figure 4: Affinity Chromatography Principles................................................................... 6
Figure 5: Fusion Protein Purification Methods................................................................... 8
Figure 6: Fusion Protein...................................................................................................... 9
Figure 7: Previously Constructed Cloned Plasmids.......................................................... 12
Figure 8: pET-ClBD-I-DEFA1 Plasmid Construction ..................................................... 13
Figure 9:.Gel Electrophoresis Check ................................................................................ 19
Figure 10: Simplified and theoretical pET-ClBD-Intein-DEFA1 Plasmid....................... 20
Figure 11: Purification Result with Different Stains ........................................................ 21
Figure 12: Purification Attempts (varying expression conditions)................................... 22
10.
11. 1
1.0 Introduction
1.1 Proteins
Proteins are long chains of amino acids with important biological functions which
exist in all living things. Some protein functions include: controlling chemical reactions
that regulate cellular function like battling disease, replicating DNA, transporting
molecules and digesting food1
. Proteins differ due to their amino acid sequences, which
control the three dimensional structure that plays a significant role in function. Proteins
are embedded within every organism’s DNA sequence and are expressed either when
necessary or when activated. These diverse roles have numerous applications which
promote research in biological functions, structure and stability of proteins2
. Since DNA
sequences are similar across a wide variety of species, the proteins synthesized also are
both chemically and physically similar, allowing for the protein of a vertebrate to be
produced in another species, such as bacteria. For human applications, purification of the
pharmaceutical protein of interest from host cells is especially important in order to avoid
an immune response. There are two main classes of pharmaceuticals today: antibody and
non-antibody peptides. Antibodies are structurally similar and can achieve high purity in
one step.
12. 2
1.2 Inteins
Inteins, intervening proteins, are sequences embedded in the host's DNA sequence
which are self-splicing protein elements. Inteins, which are analogous to introns in DNA,
should be removed for a protein to become active or mature. Several archaeal, eubacterial
and eukaryotic genes contain inteins which join the exteins together3
, (Figure 1). In order
for the intein to function properly, the intein must be in-frame with the precursor protein.
Free Inteins
N-Terminus end
(1st extein CYS)
C-Terminus end
(1st extein Ser 155)
Figure 1. Intein Function Upstream (left)
Protein Splicing: Intein joining exteins together; Upstream (left)
N-terminal extein (N-extein); C-terminal extein (C-extein)
r
Inteins
Extein-1
Inteins
Extein-3Extein-2
+ Intein Intein
Extein-1 Extein-2 Extein-33. Mature
Protein
Made
2. Splicing Step
Ligation of Exteins
1. Precursor
Protein
InteinsExtein-1 Extein-2 Intein Extein-3
13. 3
By modifying the N and C-terminal extein's amino acid (AA) sequences, the
intein behaves differently than what is seen in Figure 1, which primarily results in the
intein self-cleave or cut off of a protein section versus ligating them or connecting
exteins. These modified inteins have the ability to cleave a protein after expression,
resulting in one or two proteins, the precursor, and target protein4,5
. Inteins maintain their
ability to cleave when adjacent to non-native host proteins3
. One modified intein, ΔI-CM,
is a C-terminal cleaving, mini intein4
. This means that the N-terminus will cleave
resulting in the C-terminal extein being expressed to be produced as displayed in Figure
2.
Self-cleaving of intein resulting in the C-Extein, is a consequence
of changing the first AA from Ser155 to Ala.
Figure 2.: C-terminal Cleaving
InteinN-Extein C-Extein
InteinN-Extein C-Extein
pH or temperature shift
+
14. 4
1.3 Antimicrobial Peptides and Purification
Over the past several years, there has been a growing concern over bacterial
resistance to antibiotics, such as streptomycin becoming more resistant upon the first
application of an antibiotic, potentially resulting in multi-drug resistant bacteria7,8
.
Increasing antibiotic resistance of bacteria presents an immediate global health challenge
and has prompted the search for new therapeutics7
. This collectively results in bacteria
being unaffected by the strongest of antibiotics, penicillin9
, which then results in
antibiotic resistance. One solution is to produce new antibiotics, which is costly and
considered a short term solution since bacteria could become multi-drug resistant8
. An
alternative is the production of antimicrobial proteins that are peptide chains typically
less than 100 amino acid residues and encoded within the DNA sequences of many life
forms. The large variety of antimicrobial peptides across different species gives rise to a
large, new class of antibiotics. A subclass of these antimicrobial peptides (AMP) is called
defensins. Studies suggest that both defensin and AMP have a role in immune
response/host defense and have proven that a bacteria’s ability to gain resistance is lower
when compared to conventional antibiotics10
. This is further supported by the fact that
our human immune system contains all of these antimicrobials and to this day bacteria
have not developed any noticeable resistances to the defensins10
. There are many
setbacks to using AMPs, primarily involving production methods.
15. 5
AMPs can only be produced via chemical synthesis or through recombinant
expression, since purifying AMPs from their natural sources is too complicated. Through
chemical synthesis the active protein can be produced on a lab scale but becomes too
expensive if scaled up11
. The more cost effective solution is through recombinant
expression in bacteria, since the proteins are physically and chemically similar between
organisms, in this case E. coli. Recombinant expression involves isolating a certain DNA
sequence, α-defensin (specific AMP), and inserting it into a bacteria’s DNA sequence.
This way as the bacteria grows, or if induced (forced to express protein), it will produce
the protein of interest along with other proteins found in the bacteria’s DNA sequence, as
shown in Figure 3.
For human applications, purification of the protein of interest from the host
proteins is essential in order to avoid an immune response. Recombinant expression of
AMP does have setbacks, primarily the fact that production of these peptides would be
fatal to the cell.
Host Protein
Protein of Interest
Figure 3: Host Cell (E. coli)
Bacterium generating proteins and protein of interest
16. 6
1.4 Fusion Protein and Affinity Chromatography
One way to address the problems associated with AMP recombinant expression
involves constructing a bigger, more stable protein called the fusion protein. The use of
affinity chromatography through a fusion protein is a potential platform technology,
allowing virtually any protein to be produced. Affinity chromatography uses a polymer-
matrix that contains bio-specific sites, ligand binding domain (LBD) contains a
quarternary amine, on the resin which covalently bonds to a choline binding domain
(ClBD) affinity tag, on the fusion protein, Figure 412
.
The use of affinity chromatography for protein purification is performed on an
affinity column in cycles, and due to the specific binding of the ligand to the ClBD, better
separation of the fusion protein is obtained with fewer steps; furthermore, the nature of
the column allows for it to be reusable for a thousand-fold purifications.
The Ligand
(L),a quaternary
amine, forming
a covalent bond
between the (S)
a fusion
protein’s ClBD
Figure 4: Affinity Chromatography Principles
17. 7
There are several potential affinity tags and resins used in affinity
chromatography which can be interchangeable, varying in specific binding and binding
sites. The general disadvantage of using affinity chromatography is the initial cost of the
resin. Although reusable, overtime the resin can foul due to cell debris and chemical
degrade, affecting the target protein.
The use of an affinity tag that is genetically fused to the target protein allows for
easy purification13
. The various affinity tags differ by size and influence on the target
protein’s activity, solubility, and the binding capacity of the fusion protein to the resin,
which then requires the affinity tag to be removed. One way to remove the affinity tag is
to use proteases to remove the affinity tag for AMP purification, which is costly as well
as results in an additional processing step to separate the proteases from the target
protein14,15,
left half of Figure 5.
Another, more cost effective way, to remove the affinity tag is to incorporate a
self-cleaving intein which provides a potentially rapid and convenient method for AMP
purification, right half of Figure 5. The intein cleaves due to either a pH or temperature
shift, giving control over when the target protein is produced. A summary of the two
purification methods which implement fusion proteins is in the Figure 5 below.
18. 8
Figure 5: Fusion Protein Purification Methods
Protease based purification method (Left) and Self-cleaving Intein (Right)
19. 9
The intein cleaving reaction is limited to bacteria and difficult to control,
potentially resulting in premature cleaving. Premature cleaving is anytime the intein
cleaves before binding to the affinity column or during expression. The results of
premature cleaving is a decrease in the target protein's yield, a decrease in fusion protein
to column binding (binding capacity), and in this case the production of a toxic substance,
the antimicrobial13
. Recombinant expression of a fusion protein, Figure 6, containing an
affinity tag, self-cleaving intein, and protein of interest is a viable platform to produce
any protein, and is cost effective if the binding capacity is sufficiently high.
Ideally AMPs are desired to have a high purity, (above 80% for one pass), and
high binding capacity, (at least 50 mg/mL). AMPs have been successfully purified using
a fusion protein similar to Figure 3, which resulted in a high purity (95%) but with low
yield (<1 mg/mL)16
.
Previously, in Dr. David Wood’s research group, an attempt to purify AMP
defensin α-1 (DEFA1) via a precipitation tag, Elastin Like Peptide (ELP) was performed.
The process theoretically produced 3mg/mL DEFA1, but the activity of the peptide was
not confirmed. The low recombinant yield of DEFA1 was believed to be caused by the
large size of ELP tag, 550 amino acids (AA) in length. The larger the fusion protein the
more time needed for the fusion protein to be expressed. A larger fusion protein
Abbreviations: Affinity Tag (Choline Binding Domain)= B;
Self-Cleaving Intein (Intein) = I; α-defensin (DEFA1) = D
Figure 6: Fusion Protein
Affinity Tagα-defensin (DEFA1) Self-Cleaving Intein
20. 10
decreases expression and purification of the target protein. From ELP-intein studies, the
yield of a fusion protein can improve by decreasing the total length, which increases the
overall metabolic efficiency of the bacterial cell17
. The modified intein, ΔI-CM or I for
short, is 165 AA in length, and from previous research with Dr. David Wood, a smaller
ELP tag 200 AA in length was produced.
Assuming proper folding of the fusion protein during expression, a previously
modified ELP10 tag-fusion protein would total 83 kDa in length versus the ClBD tag-
fusion protein totaling 36 kDa in length. The ClBD tag-fusion protein is 80% smaller
than the ELP tag fusion protein, theoretically producing more ClBD tag-fusion protein
than the ELP tag-fusion protein18
. The use of the ClBD should theoretically produce a
higher yield of DEFA1 with fewer cell resources.
21. 11
2. Materials and Methods
2.1 Cloning ClBD-Intein-DEFA1 Vector
Previous work by Theodore Rader and Michael J. Coolbaugh in Dr. Wood’s lab
created a cloned plasmid with pET vector ELP-Intein-DEFA1 fusion protein and a ClBD-
Intein-maltose binding protein (MBP), respectively, as seen in Figure 7. Via a sterile
metal loop, both clones were streaked onto plates of Luria Bertani (LB) agar media with
50 mg/mL of ampicillin for 14 hours. Isolated colonies on each plate were selected and
picked to be inoculated into a 3 mL of LB broth containing 50 mg/mL of ampicillin and
were shaken at 180 RPM overnight at 37ºC (16 hours). The clones, displayed in Figure
7, containing the previously made plasmids were harvested via QIAprep Spin Miniprep
Kit (Qiagen).
22. 12
The purified plasmid DNA are then double digested with two restriction enzymes
AgeI and BamHI (New England Biolabs) and checked with 1% m/v agarose via gel
electrophoresis. Using a computer program ApE, successful digestion was checked by
comparing the theoretical bands to the experimental bands, displayed in "Results" in
Figure 9. Then the ClBD, or in this case the insert and, pET Intein-DEFA1 vector
backbone were cut out of the gel and extracted and purified by the QIAquick Gel
Extraction kit. Using a 5:1 ratio of insert to vector and T4 DNA Ligase (New England
Biolabs), the insert and vector were annealed (ligated) for 30 minutes at 0 ºC to form
pET-ClBD-Intein-DEFA1 (the expected plasmid), displayed in Figure 10. The expected
plasmid was then sent off to be sequenced. After the pET-ClBD-Intein-DEFA1 plasmid
was confirmed via sequencing it was inserted into z-competent BLR and DH5α cells.
Figure 7: Previously Constructed Cloned Plasmids
Ted's Plasmid Clone (Left); Michael's Plasmid Clone (right): Restriction site (red box)
ClBD
Tag
T7
Promoter
Ampicillin
Resistance
GeneMBP
Intein
(BamHI)
Restriction
Site
(Age I)
Restriction
Site
ELP
Tag
T7
Promoter
Ampicillin
Resistance
Gene
DEFA1
Intein
(Age I)
Restriction
Site
(BamHI)
Restriction
Site
23. 13
.
Figure 8: pET-ClBD-I-DEFA1 Plasmid Construction
+
ELP Tag (Not Used)
CBD Tag
INSERT
CBD Tag
T7 Promoter
Ampicillin
Resistance
GeneDEFA1
Intein
Ligation with T4 Ligase
+
CBD Tag
Insert
ELP Tag
T7
Promot
er
Vector
Ampicillin
Resistance
DEFA1
Intein
T7
Promoter
Ampicillin
Resistance
Gene
MBP
Intein
T7
Promoter
24. 14
2.2 Expression of Protein
The confirmed pET-ClBD-Intein-DEFA1-BLR cells were plated on LB agar
containing 50 mg/mL of ampicillin and incubated for 16 hours at 37 ºC. A single colony
was selected and used to inoculate 3 mL of LB media with 50 mg/mL ampicillin
concentration and shaken overnight at 180 RPM and 37 ºC for 14 hours. A small sample
was diluted at least 1:100 into a 50 mL solution containing 45 mL LB media and 5 mL of
Terrific Broth (TB) media as well as 50 mg/mL ampicillin for 4 hours at 37 ºC or until
the cell density OD600 was within 0.8-1.0. Once the cell density was within the range, the
50 mL solution was cooled to 16 ºC where a pre-induction sample was taken, followed
by inducing the solution to a final concentration of 1 mM Isopropyl β-D-1-
thiogalactopyranoside (IPTG) for 24 hours at 16 ºC.
After overexpression, the culture was centrifuged for 10 minutes at 5,000g and 4
ºC separating the media from cell pellets. The media was then disposed of and the cell
pellets were resuspended with 10 mL of lysis buffer or pH 8.5 1M Colum Buffer (NaCl
pH 8.5 CB) [CB is 20 mM AMPD, 20 mM PIPES, 1 M NaCl, 2mM EDTA, 1 mM DTT]
and stored at -20 ºC.
25. 15
2.3 Protein Purification of DEFA1
Prior to protein purification, the proteins must be separated from the other cellular
components. The preparation steps begin with thawing the frozen cell pellets in 8.5 pH
CB (CB is 20 mM AMPD, 20mM PIPES, 1 M NaCl, 2 mM EDTA, 1mM DTT) solution.
Once the cell pellets are resuspended in the solution, the proteins are released by
breaking, or lysing, the cell membrane which puts all the proteins in solution (assuming
the fusion protein folded properly and is soluble in the solution).
The proteins are then separated from the other cellular components by centrifuge.
After isolating the solution, the fusion protein or precursor is separated from the proteins
in solution through affinity chromatography. The affinity chromatography methods used
for purification were similar to the Intein Mediated Purification with an Affinity-Chitin
binding tag (IMPACT) system by New England Biolabs as well as other studies19
.
The exact steps and conditions used are:
(1) The frozen cell pellets were thawed, resuspended, and lysed through sonication
for 3-6 rounds, in 20 second durations, at 5W RMS or until the sample was more
transparent and the viscosity of the sample decreased.
(2) A sample was then taken called the Whole Lysate (WL)
(3) The remainder of the lysed solution was then centrifuged at 23,000 g at 4°C for
10 minutes and kept around 0°C until step (5).
26. 16
(4) A sample was taken of the supernatant or Clarified Lysate (CL) along with a cell
debris sample (CD)
(5) The lysed solution in step (3) was then diluted at about 1:5 with 40 mL 1M NaCl
8.5 pH CB.
(6) The column was equilibrated with 1M NaCl pH 8.5 CB. The affinity column
used a 3 mL Column Volume (CV) of Q-sepharose resin. After the column is
equilibrated, the diluted lysed solution from step (5) was poured carefully to avoid
suspending the resin. The column had a flow rate of 0.5 mL/min where a sample
was taken every full cycle, or Flow Through (FT), for a maximum of three flow
throughs.
(7) After the last FT, a column bed sample was taken of the resin before the cleaving
reaction. A 1 M NaCl pH 6.5 CB shift is performed to start cleaving of the fusion
protein and was run at RT for 24 hours.
(8) After 24 hours another resin sample was taken and the column is eluted for 24
hours.
(8) After 24 hours another resin sample is taken and the column is eluted with 3
Column Volumes or 9 mL of 1 M NaCl pH 6.5 CB at Room Temperature and
collected in fractions.
(9) These Eluted (E) samples are then analyzed with SDS-PAGE running buffer on a
15% acrylamide gel before and after purification.
(10) Afterwards a Tricene gel should be used to observe the small peptide.
27. 17
2.4 Defensin Activity via Radial Diffusion Assay
From a previous study, the activity of the purified DEFA1 is tested by a Radial
Diffusion Assay20
. Radial Diffusion Bacteria were grown overnight in 20 mL Tryptic Soy
Broth (TSB) for 16 hours. The overnight culture was then diluted out 1:100 (50μL) into
fresh 50 mL TSB and shaken at 180 RPM for 2.5 hours at 37°C or until mid-logarithmic
phase was achieved. The incubated bacteria were then centrifuged at 900 g for 10
minutes at 4°C. The cell pellets were then washed with pH7.4, cold 10 mM sodium
phosphate buffer (NAPB) and resuspended in 10 mL of cold NAPB. Next, a 1mL optical
density (OD) measurement around 620 nm is taken. Based on the OD620 to column
forming-unit (CFU) relationship (0.20 = 5 x 107
CFU/mL) 1 x 106
bacterial CFU was
added to 10 mL of 10 mM NABD at 42°C containing 3 mg of TSB medium, 1% w/v of
low-electroendosmosis type agarose (SeaKem), and to a final concentration of 0.02% v/v
Tween 20 (Sigma), which was previously autoclaved. The bacteria-agar mixture was
vortexed, then poured into a 100x15 mm Petri dish (Fischer Scientific) to a depth around
1 mm. The agarose layer was punctured with a template grid and 3-mm biopsy punch to
make 16 evenly spaced wells. Next 5 μL of test mixture, containing purified DEFA1, was
added to each of the wells and incubated at 37°C. After 3 hours, the gel was overlaid with
10 mL sterile, warm (42°C), NAPB double-strength containing TSB (6% w/v) and 1%
w/v
28. 18
agarose and incubated at 37°C for 20 hours. The resulting inhibition zone around each
well was examined to determine the zone of clearance.
Then the gels were stained for 24 hours in dilute Coomassive brilliant blue (2 mg
dye, 27 mL methanol, 63 mL water and 15 mL 37% formaldehyde) to aid in visual
identification. The aqueous solution in each well was decanted and replaced with a 10%
acetic acid and 2% dimethyl-sulfoxide (DMSO) for 10 minutes. This solution was
discarded and the gels allowed to dry for 2 hours. The plates were then stored for future
use. The activity of DEFA1, water, a 0.1 mg/mL lysozyme control and buffer solution
(0.5 M NaCl pH 6.5 CB) were tested against E. coli. A quantitative analysis on DEFA1
activity is based on comparing the control's circle to the purified DEFA1 circle.
29. 19
3. Results and Discussion
3.1 Creation of pET-ClBD-I-DEFA1
An expression vector consisting of the choline binding domain (ClBD) fused with
the intein (I) and the DEFA1 peptide was constructed. Several restriction sites were
chosen but resulted in too small of a DNA fragment which would not appear on the gel or
incomplete digestion (smearing of band). If the experimental gel check was consistent
with the theoretical bands, comparison in Figure 9, then successful digestion was
achieved. The bands of interest (labeled Vector and Insert) are cut out of the gel, (right)
portion of Figure 7, and annealed together to produce pET-ClBD-I-DEFA1 displayed in
Figure 10.
Figure 9:.Gel Electrophoresis Check
Theoretical Gel Check (Left); Experimental Gel Check (Right)
DNA
Ladder
(vector)
ClBD Tag
(Insert)
ELP tag
(5000 bp)
(1500 bp) (500 bp)
ELP
tag
1815 bp
Vector
5920 bp
Insert
ClBD
Tag
462 bp
Incomplete
Digestion
1. 2. 3. 4. 6. 7. 8.
8.
30. 20
Theoretical (ApE) Based Plasmid
Figure 10: Simplified and theoretical pET-ClBD-Intein-
DEFA1 Plasmid
T7
Promoter
Ampicillin
Resistance
Gene
DEFA1
Intein
ClBD Tag
Sequenced Confirmed Plasmid
31. 21
3.2 Protein Purification
After the vectors were created, purification attempts were performed. The fusion
protein is roughly 36 KDa containing: the ClBD (12KDa), Intein (19 KDa) DEFA1 (4
KDa), and other linking pieces. The first protein purification attempt, shown in Figure 11,
displays an undesired result.
Usually this trial would be ruled out before purification but the results were
expected to be favorable, and so this check was overlooked. To ensure that this result was
not due to human error, the cells were re-grown and expressed at varying conditions.
Figure 11: Purification Result with Different Stains
(Fusion protein size 36KD): Lane1- Protein Ladder, ; Lane 2-Whole Lysate (WL);
Lane 3-Cell Debris (CD); Lane 4&5- Clarified Lysate (CL), Lane 6-Flow Through
(FT 1) , Lane 7-FT2
32. 22
The varying conditions consisted of lowering the temperature and IPTG
concentration to slow down expression. Theoretically, this would result in slower folding
and promote proper protein folding. The results of varying the expression conditions are
displayed in Figure 12, which displays similar results to Figure 11, concluding that the
fusion protein is insoluble.
Figure 12: Purification Attempts (varying expression conditions)
IPTG concentration 1/10th of Figure 9 as well /as expression at 15°C
Sonication rounds doubled to insure all cells are lysed
33. 23
4. Conclusions
The investigation into shortening the affinity tag did not produce the expected
results, i.e. increased DEFA1 yield. The changing of the affinity tag resulted in improper
folding of the fusion proteins making the protein aggregate together and thus become
insoluble. This result displays how the fusion protein's components influence the
production of DEFA1. There are many possible causes as to why the fusion protein
became insoluble, such as the need for another protein during expression to ensure proper
folding. An exact explanation is not possible, although a couple of solutions are viable.
There are two possible pathways to resolve this, which are suggested for future work.
One pathway involves further altering of the fusion protein by changing the size of the
affinity tag to a larger size, such as the ELP tag, which has been shown to increase
solubility in previous studies18
. Increasing the size of the affinity tag and fusion protein
will slow down overall protein production during expression, possibly promoting proper
folding as well as making the fusion protein overall more soluble. The second viable
solution is to alter the species of the bacterium during transformation. Changing the
bacterium species to one that produces protein more slowly and promotes proper protein
folding, in addition to possibly containing enzymes necessary for proper protein folding.
In summary, the purification of DEFA1 via fusion protein utilizing a smaller
affinity tag (ClBD) was unsuccessful in the production of DEFA1, and consequently in
increasing yield. A smaller tag results in improper folding possibly suggesting a limit to
34. 24
the size of the fusion protein used for AMP production. Increasing the size of the fusion
protein, or changing the bacterium species, may be a viable solution to DEFA1
purification.
35. 25
Work Cited
1. Pierce, B. A. (2005). Genetics: A conceptual approach. NY: W.H. Freeman.
2. National Human Genome Research Institute, National Institutes of Health, & U.S.
Department of Health and Human Services. (2012, June 8). Chromosomes.
Chromosomes Fact Sheet. Retrieved March 25, 2013, from
3. Perler FB, Davis EO, Dean GE, Gimble FS, Jack WE, et al. (1994) Protein splicing
elements: inteins and exteins–a definition of terms and recommended
nomenclature. Nucleic Acids Res 22: 1125–1127.
4. Fong, B. A.; Wu, W. Y.; Wood, D. W., The potential role of self-cleaving purification
tags in commercial-scale processes. Trends in biotechnology 2010, 28 (5), 272-9.
5. Pietrokovski S. 2001. Intein spread and extinction in evolution. Trends Genet
17(8):465-72.
6. Wood, D., Derbyshire, V., Wu, W., Chartrain, M., Belfort, M., & Belfort, G. (2000).
Optimized Single-Step Affinity Purification with a Self-Cleaving Intein Applied to
Human Acidic Fibroblast Growth Factor. Biotechnology Progress, 16(6), 1055-
1063.
7. Demerec, M. "Origin of Bacterial Resistance to Antibiotics." Journal of Bacteriology.
56.1 (1948): 63-74.
36. 26
8. News, CBC. "Antibiotics Useless for Most Sinus Infections." CBCnews. CBC/Radio
Canada, 21 Mar. 2012. Web. 28 Sept. 2012.
9. Alanis, Alfonso. “Resistance to Antibiotics: Are we in the Post-Antibiotic Era?”
Review Article Archives of Medical Research, Volume 36, Issue 6, November–
December 2005, Pages 697-70.
10. Hancock, R. E.; Sahl, H.-G., Antimicrobial and host-defense peptides as new anti-
infective therapeutic strategies (Review). Nature Biology 2006, 1551-1557.
11. Chino, Naoyoshi, Shigeru Kubo, Hideki Nishio, Yuji Nishiuchi, Masamitsu Nakazato,
and Terutoshi Kimura. "Chemical Synthesis of Human Ø-Defensin (hbd)-1, 2, 3
and 4: Optimization of the Oxidative Folding Reaction." International Journal of
Peptide Research and Therapeutics. 12.3 (2006): 203-209.
12. Affinity Chromatography: Principles and Methods. Uppsala Biosciences, 2002.
13. Wood, D., 2003. Simplified protein purification using engineered self-cleaving affinity
tags. J. Chem. Technol. Biotechnol. 78, 103–110.
14. Li, Y., Recombinant production of antimicrobial peptides in Escherichia coli: a
review. Protein expression and purification 2011, 80 (2), 260-7.
15. Fong, B. A.; Wu, W. Y.; Wood, D. W., The potential role of self-cleaving purification
tags in commercial-scale processes. Trends in biotechnology 2010, 28 (5), 272-9.
16. Wright, O.; Yoshimi, T.; Tunnacliffe, A., Recombinant production of cathelicidin-
derived antimicrobial peptides in Escherichia coli using an inducible autocleaving
enzyme tag. New biotechnology 2012, 29 (3), 352-8.
37. 27
17. Meyer, D. E.; Chilkoti, A., Quantification of the effects of chain length and
concentration on the thermal behavior of elastin-like polypeptides.
Biomacromolecules 2004, 5 (3), 846-51.
18. Rader, Theodore S. "Effect of Elastin-Like Polypeptide Tag Length on the Purification
of Antimicrobial Peptides." Thesis. The Ohio State University, 2012. Kb.osu.edu.
The Ohio State University, 19 July 2012. Web. 7 Feb. 2013.
19. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence, LM, Perler FB, Benner
J, Kucera RB, Hirvonen CA, Pelletier, JJ, Paulus H and Xu MQ, Single-column
purification of free recombinant proteins using a self-cleavable affinity tag derived
from a protein splicing element. Gene 192:271–281 (1997).
20. Lehrer, Robert, Michael Rosenman, Sylvia Harwig, Richard Jackson, and Patricia
Eisenhauer. "Europe PubMed Central." Ultrasensitive Assays for Endogenous
Antimicrobial Polypeptides. Elsevier Science Publishers, 21 Mar. 1991. Web. 18
Feb. 2013.