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Facilitating unambiguous NMR assignment by solid–state NMR using segmental isotope labeling through split-inteins

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Facilitating unambiguous NMR assignment by solid–state NMR using segmental isotope labeling through split-inteins

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Facilitating Unambiguous NMR Assignment
by Solid–State NMR Using Segmental Isotope
Labeling Through Split-Inteins.

By
Samridhi Garg

August 2019

A Thesis Presented to the Faculty of the University of
Southern California – Graduate School

In Partial Fulfillment of the

Requirement for the Degree of

Master of Science

Biochemistry and Molecular Medicine
Acknowledgments:

I would like to express my sincere gratitude to Dr. Ansgar Siemer for providing me
constant encouragement and guidance while I worked as a master’s student in his lab. I
will sorely miss working in such a positive lab environment.

I would like to thank my committee members Dr. Ralf Langen and Dr. Julio Camarero for
their time and insightful questions.

I would like to acknowledge Silvia Cervantes for her careful review of my thesis. She has
always been there more as a friend than a scientist. I would like to thank all the members
of Siemer lab; Maria Soria, Alexander Falk, Connor Hurd, Shruti Bendre, Manjima Sarkar,
Jina Kim, and Rajashree Venkatraman for creating wonderful and the supportive lab
environment.

I would also like to thank Monica Pam and Dr. Judd rice who provided me invaluable
support during my journey.

Lastly, but most importantly, I would like to thank my friends and family for their love and
encouragement.

Table of Contents
1. Abstract: ......................................................................................... 1
2. Introduction .................................................................................... 2
2.1 Amyloid Fibrils: ...................................................................................... 2
2.2 Functional Amyloid ................................................................................. 3
2.3 CPEB Prion and Long-Term Memory Formation: .............................................. 3
2.4 Functional Amyloid Orb2: ......................................................................... 3
2.5 Challenges of structural elucidation ............................................................ 4
2.6 What are inteins? ................................................................................... 5
2.7.1 Mechanism of Protein Splicing: ................................................................................... 5
2.8 Segmental Isotopic Labeling by Split-inteins ................................................... 7
3. Results: .......................................................................................... 8
3.1 Protein Expression and Purification ............................................................. 8
3.2 In-vitro Trans-splicing Reaction .................................................................. 9
3.3 Troubleshooting Trans-splicing Reaction Product Purification: .......................... 10
3.3.1 Purification of Ligation Product Using Ni-NTA Beads ......................................................... 10
3.3.2 Ligation Product Purification Using Dialysis ................................................................... 11
3.3.3 Ion Exchange Chromatography ................................................................................. 12
3.3.4 Size exclusion chromatography ................................................................................. 14
3.4 Expression and Purification of Intein linked Orb2A_1- 320: .............................. 15
3.5 Mass Spectrometry ................................................................................. 16
3.6 Expression and Purification of Non-ligated Orb2A_1- 320: ................................ 17
3.7 Intrinsically Disordered Orb2A_320 Protein Can Accumulate in Droplet- like
Assemblies ................................................................................................ 20
3.8 Analysis of Amyloid Fibrils by Electron Microscopy ......................................... 20
3.9 Thioflavin T Assay: ................................................................................. 22
4. Discussions: ................................................................................... 24
5. Materials and Methods: .................................................................... 26
5.1 Plasmid Design ...................................................................................... 26
5.2 Transformation and Protein Expression: ...................................................... 27
5.2.1 Transformation ............................................................................................. 27
5.2.2 Protein Expression ......................................................................................... 27
5.3 Cell Lysis ............................................................................................. 28
5.3.1 For Orb2A NTD1-88 fused intein Segment ............................................................... 28
5.3.2 For Orb2A CTD89-540 Fused Intein Segment (Inclusion Body Purification) .......................... 28
5.3.3 Cell Lysis of Non-ligated Orb2A_320 (DNase Treatment) .............................................. 29
5.4 Purification: ......................................................................................... 29
5.5 Troubleshooting Trans-splicing Reaction Product Purification: .......................... 31
5.5.1 Purification of the Reaction Product Using Ni-NTA column ........................................... 31
5.5.2 Purification of the Reaction Product Using Dialysis .................................................... 31
5.5.3 Purification of the Reaction Product Using Ion Exchange Chromatography ......................... 31
5.5.4 Purification of the Product Using Size Exclusion Chromatography ................................... 32
5.6 UV spectroscopy: .................................................................................. 32
5.6.1 UV 280 nm .................................................................................................. 32
5.6.2 Fluorescamine Assay ...................................................................................... 32
5.7 Formation of Droplets and Amyloid Fibrils Assemblies: .................................... 33
5.8 Transmission Electron Microscopy .............................................................. 33
5.9 Thioflavin T staining ............................................................................... 33
6. References: .................................................................................... 35

1
1. Abstract:
Solid-state NMR has become a powerful tool for studying the structure, dynamics and
interaction of supramolecular protein assemblies under near physiological conditions. The
major limitation of protein NMR is its spectral crowding resulting in resonance overlap,
which increases with the number of residues. Segmental isotope labeling allows specific
segments within the protein to be identified by NMR and thus reduces the spectral
complexity. In my thesis, I will present my work on the split intein DnaE from Nostoc
punctiforme to apply sparse isotope labeling strategies to the functional amyloid Orb2A.
Split-inteins are protein introns that splice out autocatalytically joining the target protein
by peptide bond. We presented an efficient method for production of the segmentally
labeled Orb2A in which either the N or C terminal domain is uniformly labelled with
15
N,
13
C. The NTD of Orb2A was fused with the NTD of Npu DnaE (NTD1-88 fused intein)
and its CTD was fused with the CTD of Npu DnaE (CTD88-540 fused intein). We
observed high-yield the intein fusion proteins. Additionally, robust trans-splicing with an
efficiency of more than 90% splicing product was obtained. However, we observed
difficulty in separating ligation products from the educts because the original idea of
separating ligation product from Ni-NTA column failed due to the presence of histidine
domain in the intein-linked Orb2A. We utilized different purification strategies such as
dialysis, ion exchange chromatography, and size exclusion chromatography, however,
none of them were able to separate intein-linked orb2A from the educts; the intein-linked
Orb2A and the educts were eluted together in all the purification techniques mentioned
above. Ultimately, adding tween in the reaction buffer aided in separating ligation product
from the educts.
2
2. Introduction
2.1 Amyloid Fibrils:
Amyloid fibrils are insoluble, proteinaceous deposits that are rich in β-sheet structure,
exhibit fibrillar morphologies, and can form under certain conditions in-vivo and in-vitro
(1). Their formation can accompany a range of neurodegenerative disorders including
Alzheimer’s diseases (AD), Parkinson’s disease (PD), and Huntington’s diseases (HD)
(2). In AD, for example, aggregates of amyloid beta form plaques that can be found in the
brain tissue of Alzheimer's patients and which are thought to block cell to cell signaling at
the synapse. Amyloid fibril formation is also associated with other diseases, such as type
II diabetes and Creutzfeldt-Jakob disease.

A wide range of studies have revealed information about the structure of the amyloid fibrils
at different levels. Structural definition of an amyloid fibril is an unbranched protein fiber
whose repeating substructure consists of β strands that run perpendicular to the fiber
axis, resulting in a cross-β sheet (1,3). Several microscopes such as transmission
electron microscopy (TEM), atomic force microscopy (AFM) and scanning electron
microscope are effective in imaging amyloid fibrils in vitro (see Fig. 1). The images
associated with electron microscopy reveal that amyloid fibrils are long, unbranched
filaments with a diameter of 6-12 nm. The constituent units of amyloid fibrils are formed
from 2-6 unbranched protofilaments (20–30 A˚ in diameter) that twist together or
associated laterally to form fibrils (1,4). Recent progress in X-ray crystallography, Nuclear
Magnetic Resonance (NMR), Cryo-electron microscopy and Electron Paramagnetic
Resonance (EPR) have contributed to the structural characterization of amyloid fibrils at
the molecular level.

Figure 1: Structural model of amyloid fibrils. (A) image of the amyloid fibril by transmission electron
micrographs. (B) Schematic illustration of the cross-□ structure in protein aggregates, with the hydrogen
bonds represented by dashed line. (C) X-ray fiber diffraction pattern of a cross-□ sheets in the fibrils.
Image from Greenwald, Jason, and Roland Riek. “Biology of Amyloid: Structure, Function, and
Regulation.” Structure, vol. 18, no. 10, 2010, pp. 1244–1260., doi: 10.1016/j.str.2010.08.009.
3
2.2 Functional Amyloid:
Amyloid fibrils are commonly associated with a loss of function in the cell and therefore,
initially believed to be detrimental to humans. However, a growing number of evidences
indicates that amyloid fibrils can be beneficial. For example, in humans, the functional
amyloid such as Pmel17 plays an important role in the biosynthesis of human melanin
pigment (5). Similarly, amyloids in fungi such as Aspergillus produce hydrophobins to
penetrate the air-water interface (31). In bacteria such as Escherichia coli
and Salmonella spp., the functional amyloid called curli, contributes to host cell adhesion
and invasion, biofilm formation and immune system activation (6). The functional
amyloid cytoplasmic polyadenylation element binding proteins (CPEBs) in fruit fly
Drosophila, Aplysia californica, and mice are essential for long-term memory formation.

2.3 CPEB Prion and Long-Term Memory Formation:

In order to understand the mechanism behind long-term memory formation, it is very
important to understand how long-term memory persists when proteins that initiate the
process degrade over time. Synaptic plasticity and synaptic potentiation play a very
important role in the long-term memory formation. For long-term memory, long lasting
forms of synaptic plasticity such as long-term facilitation (LTF) and long-term potentiation
are required. Post-translation modification of the new proteins regulates these forms of
synaptic plasticity. Recent research is focused on understanding transcriptional regulation
at the synapse. The family of prion-like protein CPEBs is one such translational regulator.
CPEB proteins are the RNA-binding proteins that when aggregated, promote the mRNA
polyadenylation of the specific protein at the synapse. CPEB proteins are expressed in
neurons and bind to the U-rich cytosolic polyadenylation elements (CPEs) of mRNA 3’-
UTR and therefore polyadenylate these mRNA (9). Activation of CPEB allows elongation
of poly-A tails of CPE-containing mRNA, which allows the expression of synaptic specific
proteins. For example, in Xenopus, CPEB promotes the polyadenylation-induced
translation of dormant mRNAs depending on the stage of development. Furthermore, a
CPEB isoform has been identified in the nervous system of Aplysia (apCPEB), which
forms amyloid-like aggregates that are essential for the maintenance of long-term
synaptic facilitation (8). In mice, deletion of CPEB3 gene reduces hippocampal synaptic
plasticity and long-term memory formation. In Drosophila, amyloid-like aggregates of Orb2
activate the mRNA polyadenylation, which allows the synaptic-specific protein expression
(10). These observations suggest that CPEBs plays an important role in regulating and
maintaining long-term memory formation.

2.4 Functional Amyloid Orb2:

The functional amyloid Orb2 has two isoforms, Orb2A and Orb2B, which differ in their N-
termini (11). The C-terminus, which is composed of two RNA recognition motifs (RRM)
and a Zinc finger, is the same in both isoforms (see Fig. 2). Both isoforms share a
common glutamine-rich domain, which is comparable with the poly-glutamine (poly-Q)
domain found in several proteins responsible for neurodegenerative diseases.
4
Figure 2: Domain structure of Orb2B and Orb2A. Q is the glutamine rich
domain, G is the glutamine rich domain, RRM is the RNA recognition motif, ZN
is the Zn
+2
fingers.
Furthermore, a similar poly-Q domain was observed in the prion forming domain of
apCPEB (27). The N- terminus of Orb2A is much shorter, as compared to the longer
isoform Orb2B, and contains 8 amino acids unique to this isoform. In contrast, Orb2B has
162 amino acids ahead of the prion-like domain. The biological activity for the two isoforms
is also different and Orb2A, the less abundant of two, is more efficient at activating fibril
formation both in vitro and in vivo (28,11). Additionally, a point mutation in Orb2A,
Orb2AF5>Y5, reduces the fibril formation of Orb2B and is therefore believed to be
responsible for seeding the fibril formation (28). Previous studies have shown that Orb2A
functions without its C-terminal RRMs l (28,29).

2.5 Challenges of structural elucidation:
Traditionally transmission electron microscopy (TEM) and atomic force microscopy are
used in the identification of amyloid polymorphisms. Yet, determining the high-resolution
structural characteristics of the fibrils are very challenging. The inheritably non-crystal
forming structure of the amyloid fibril makes them a poor candidate for X-ray
crystallography (19). Liquid state NMR is used to characterize amyloid proteins in the
soluble state and determine the amyloid forming regions of the proteins. However, the
slower tumbling of the molecules due to their large size precludes structural elucidation
by liquid state NMR (19). Alternatively, solid-state nuclear magnetic resonance (ssNMR)
is a highly appreciated high-resolution structural determination method for the amyloid
proteins.

However, as the molecular weight of the NMR sample increases, successful assignment
of the NMR resonances becomes difficult due to spectral congestion and resonances
overlap. Advances in the development of sophisticated pulse schemes, as well as the
introduction of selective isotope labeling strategies, have addressed a number of these
5
challenges. The uniformly labeled
13
C,
15
N-labeled samples hold the most possible
information, however, the microheterogeneity of amyloid structure results in the spectral
overcrowding and assignment ambiguities (18). Therefore, segmental isotope labeling
strategies have been employed; these rely on uniformly
15
N,
13
C labeling of either the N-
terminal domain or the C- terminal domain. One approach to achieve sparse isotope
labeling is through a trans-splicing reaction using the DnaE split intein (17).

2.6 What are inteins?
Inteins are the auto processing domains that mediate a traceless protein ligation process
known as protein trans-splicing (PTS) (21). These proteins are known as protein intron.
Like introns, they excise themselves out from the host polypeptide through the cleavage
of the peptide bond and, in this process, joins the remaining portion of the protein. These
functions are controlled by the intein amino acid sequence and protein sequence flanking
the intein. Intein mediated protein splicing is encoded by a small fraction (less than 5%)
of intein genes (22). Protein trans-splicing reaction occur after the intein genes are
transcribed and translated into precursor proteins, which contain three segment– intein,
N-extein (host protein sequence preceding the intein) and C-extein (sequence following
intein known) (24). Inteins were first discovered 25 years ago through the gene sequence
analysis, which showed that the extra sequence of Saccharomyces cerevisiae Sce VMA1
vacuolar ATPase gene was removed after a translation from the host protein (21). Inteins
are found in all eukaryotes, bacteria, archaea, and viruses. According to the intein
database (Inbase), there are more than 350 inteins present in all three domains of life
with 70 inteins in eukaryotes, 150 inteins in eubacteria and 110 inteins in archaea (22).
The inteins with a minimum length of 134 amino acids (mini-inteins) and a maximum length
of 844 amino acids are present in the database (22). Sometimes, splitting the intein domain
into two polypeptide chains facilitates the linkage of the flanking amino acid sequences.
The intein involved in this process is known as split-inteins, which is encoded by two
different genes (23). The first split-intein sequence was published in cyanobacteria DnaE,
where two separate genes, dnaE-n and dnaE-c, encoded the catalytic subunit α of DNA
polymerase III (26).

2.7.1 Mechanism of Protein Splicing:
Intein mediated splicing is a four-step process (see Fig. 2). First, the nucleophile (cysteine
or serine) attacks the preceding peptide bond linking the N-extein and intein resulting in
the formation of the thioester bond (Step 1). Second, the nucleophile at the start position
of the C-extein (cysteine, serine or threonine) attacks the N-extein by transesterification,
resulting in the formation of branched ester intermediate in which N-extein and C-extein
are attached (Step 2). Third, the asparagine cyclization cleaves the peptide bond between
the intein and the C-extein facilitating the release of free intein from the exteins (Step 3).
Finally, the ester bond containing the N-extein and C-extein is rapidly converted to an
amide bond resulting in a rearrangement to form a stable peptide bond in an intein-
independent fashion. (step 4). (25)
6
2.8 Segmental isotope labelling by Split-inteins.

Figure 2: Schematic representation of the mechanism of protein splicing. It is a four-
step mechanism as shown in the figure. Image taken from Mills, Kenneth V et al.
“Protein splicing: how inteins escape from precursor proteins.” The Journal of
biological chemistry vol. 289,21 (2014): 14498-505. doi:10.1074/jbc.R113.540310
7

2.8 Segmental Isotopic Labeling by Split-inteins:
One of the most important applications of protein ligation by split-intein is segmental
isotope labelling, where only one section of the amino acids chain (usually either the N-
or the C- terminal sections) is labelled for the NMR spectroscopy studies. NMR is a very
powerful tool for elucidating the 3D structures under near physiological conditions. Protein
of interest in NMR studies requires stable incorporation of
15
N,
13
C, and/or
2
H nuclei in the
backbone and side chains (34). In general, growing recombinant proteins in an
isotopically supplemented medium help to incorporate these isotopes uniformly because
the natural abundances of these isotopes are low (34). The application of the large
protein NMR is limited by the high molecular weight and the resulting spectral
congestions. Using a protein splicing reaction with split-inteins allows to label a segment
of the protein and ligate it to generate a segmentally labeled full-length protein sample
with a simplified NMR spectra. In the following section I will describe my effort to
segmentally label the Orb2A using the split- intein technology.

Figure 3: Schematic representation of the N- and C- terminal halves of the Orb2A
fused with the N- and C- terminal domain of the intein.
3. Results:

3.1 Protein Expression and Purification
For segmental isotope labeling, Orb2A was divided into two parts (i) the N-terminal
domain (NTD) (amino acids 1-88) and (ii) the C-terminal domain (CTD) (amino acids 88-
540). The NTD of Orb2A was then fused with the NTD of Npu DnaE (NTD1-88 fused
intein) and its CTD was fused with the CTD of Npu DnaE (CTD88-540 fused intein) (see
Fig. 3)

We started expressing and purifying the intein fusion proteins. We purified the NTD1-88
fused intein using denaturing conditions and refolded it on the column using renaturing
buffer. Purification using Ni-NTA beads resulted in pure NTD1-88 fused intein, according
to SDS-PAGE analysis (See Fig. 4). We used an imidazole step gradient to elute the
protein. The majority of the protein eluted at 150mM imidazole.

8
Figure 4: SDS-PAGE (14%) analysis of the purified NTD1-88 fused inteins. The
majority of the impurities are removed in NaCl wash. While the majority of pure
fraction is eluted in 100mM and 150mM.

9
Figure 5: SDS-PAGE (14%) analysis of the purified CTD89-540 fused intein. The
majority of the impurities are removed in Triton-X 100 wash. While the majority of pure
fraction is eluted at pH 3.75.
Similarly, we expressed and purified CTD89-540 fused intein from inclusion bodies
produced in E.coli. The purification was done using Ni-NTA beads and elution was done
using a pH gradient. The purification was done using Ni-NTA beads and elution was done
using a pH gradient. The majority of the CTD89-540 fused intein eluted at pH 3.75 and
the procedure resulted in relatively pure protein according to SDS-PAGE analysis (see
Fig. 5).

For both intein fusion proteins most impurities were successfully removed in the flow-
through and washes (Triton-X, NaCl and pH 6.75). After comparing the SDS-PAGE gels
for the purification of NTD and CTD inteins fusion proteins, we observed that the yield of
NTD1-88 fused intein (dark band) was higher in comparison to the CTD89-540 fused
inteins (lighter band).

3.2 In-vitro Trans-splicing Reaction
We restored the trans-splicing reaction activity of the intein fused proteins by exchanging
the reaction mixture against a buffer with a lower concentration of urea. For this we used
PD-10 desalting columns. Optimal results for the trans-splicing reaction were achieved in
the presence of 4M urea. We monitored the progress of the trans-splicing reaction using
SDS-PAGE and observed that almost 90% of the splicing product was obtained within 10
minutes of incubation at room temperature. No further changes were observed after 24
hours in the trans-splicing reaction (see Fig.6)
10

3.3 Troubleshooting Trans-splicing Reaction Product
Purification:

3.3.1 Purification of Ligation Product Using Ni-NTA Beads
Next, we purified intein-linked Orb2A from the educts (NTD1-88 fused intein and inteins).
The original idea was to purify the ligation mixture using Ni-NTA beads since the unligated
precursor protein and the inteins have 6xHis tags and the product does not. However, the
original idea failed because NTD of intein-linked Orb2A has a histidine rich domain in its
sequence, which allows it to bind the Ni-NTA beads as well (see Fig. 7). To solve this
problem, we tried using a lower concentration of imidazole (20 mM) to elute Orb2A which
should be loosely bound to the Ni-NTA beads since it contains 4xHis in comparison to the
6xHis tag on the educts (CTD89-540 fused intein and intein).Yet in the SDS-PAGE
analysis, we observed two bands at 20 mM imidazole concentration, which corresponded
to the Orb2A and CTD89-540 fused intein (see Fig.8).

Figure 6: SDS-PAGE of the time-course for the protein ligation reaction of NTD1-88 fused
intein and CTD89-540 fused intein in the presence of 4M urea, 250mM NaCl, 50mM Tris,
11

3.3.2 Ligation Product Purification Using Dialysis
As a next attempt to separate the ligation products, we used dialysis. The reasoning
behind this was that there was a considerable difference between the molecular weights
of Orb2A (MW 58 kDa), NTD1-88 fused intein (MW 26 kDa), and inteins (MW 11 kDa).
Therefore, a dialysis membrane with a 30 kDa molecular weight cut off was used. The
idea was that the ligation product, whose molecular weight was greater than 30kDa, would
remain in the dialysis membrane. While on the other hand the educt, whose molecular
weight was less than 30 kDa, would leave the membrane. However, it turns out that the
educts, with a molecular weight less than 30 kDa, didn’t leave the dialysis membrane (see
Fig. 9)
Figure 7: schematic
representation of the Sequence of
the N-terminus of Orb2A
Figure 8: SDS-PAGE analysis of the
purification step on Ni-NTA beads. While
some of the ligation product and the
educts were isolated in the Flow-through
and the 150mM Imidazole elution. The
majority was isolated in the 20mM
Imidazole elution.
12

3.3.3 Ion Exchange Chromatography
Next, we attempted to use cation exchange to separate Orb2A from the educts (NTD1-
88 fused intein and intein) because there was a difference between the theoretical
isoelectric point (pI) of educts (NTD1-88 fused inteins = 5.81 and inteins = 5.52) and
product = 7.65. We equilibrated the column at pH 6.5 and a salt gradient was used to
elute. Product, which was negatively charged, should bind the resin while educts, which
should be positively charged, should not bind the resin. However, what we found was that
both the products and educts bound the cation exchange resin (see Fig. 10,11). In
following attempts, we equilibrated the cation exchange resin at pH 6.0, pH 7.0 and pH
7.5 respectively, however, the educts still bound the resin.
Figure 9: 14% SDS-PAGE analysis of the purification step using dialysis membrane
(30kDa). The educts (NTD1-88 fused inteins and the inteins) whose molecular weight
were less than 30kDa were not observed in the dialysate (Lane 4 and 5). However,
Lane 6 represents the concentrated product along with concentrated educts
13
Figure 10: Ion exchange chromatogram of the ligation mixture after 24 hrs of
incubation. UV absorption peaks are marked as peak1, peak2, and peak 3. Peak 1
and peak 2 are below the detection limit. Peak 3 correspond to the mixture of the
ligation product and educts on the SDS-PAGE (NTD1-88 fused inteins and inteins)
Figure 11: 14% SDS-PAGE analysis with Zinc Stain of the Cation exchange
chromatogram peaks. While peak 1 and peak 2 are below the detection limit. Different
fraction of peak 3 aliquots corresponds to the both ligation product and the educts

14
Figure 12: Size exclusion chromatogram (sephacryl S-200 column) of the ligation
mixture after 24 hrs of incubation. UV absorption peaks are marked as peak1,
peak2, peak 3, and peak 4. Peak 1 and peak 2 are below the detection limit. Peak
3 and peak 4 correspond to the mixture of the ligation product and educts on SDS-
PAGE (NTD1-88 fused inteins and Inteins)
3.3.4 Size exclusion chromatography
We then decided to use size exclusion chromatography to purify intein-linked Orb2A from
the trans-splicing reaction mixture since there is a significant size difference between
product (MW = 58kDa) and educts (NTD1-88 fused inteins MW 26 kDa and inteins MW
11 kDa). A sephacryl S-300 column was used for the purification, however, the ligation
product and the NTD1-88 fused inteins were not separated. Instead, they both came out
together in one peak. To get better resolution, we tried a sephacryl S-200 column,
however the product and the educts were not separated and eluted in all peaks in size
exclusion column chromatogram (see Fig. 12,13).

Figure 13: 14% SDS-PAGE analysis of the size exclusion chromatography peaks.
Peak 1 was below the detection limit. Different aliquots of peak 2 and peak 3
correspond to the mixture of the product and the NTD1-88 fused inteins. Peak 4
aliquots correspond to the mixture of intein and product.
15
3.4 Expression and Purification of Intein linked Orb2A_1- 320:
In order to improve the yield of the CTD89-540 fused intein, we fused Orb2A CTD (amino
acids 89-320, without the RRMs) with inteins (CTD89-320 fused intein). The reasoning
behind this was that Keleman and coworkers showed that Orb2A without CTD RRMS
leads to LTM similar to the full-length Orb2A (28). We expressed Orb2A CTD (89-320)
fused with CTD of Npu DnaE (CTD89-320 fused intein) in BL-21 E.coli strain and purified
them in inclusion bodies using Ni-NTA beads. Gel electrophoresis results showed a better
yield of CTD89-320 fused intein which in turn helped in improving the yield of the ligation
product. Although we improved our yields, we still observed similar problems to those of
full length Orb2a when purifying the ligation product. We hypothesized that perhaps there
was a non-specific interaction between the ligation products and educts (unligated
precursor protein and cleaved inteins segment).

Based on this hypothesis, we added 500mM NaCl and 0.1% tween to the reaction buffer
to discourage non-specific interactions. The trans-splicing reaction time was also kept at
1 hour. We purified the ligation product using a sephacryl s-200 column and observed
three UV absorption peaks on the size exclusion chromatogram. According to SDS-PAGE
analysis these peaks correspond to Ligation product (Orb2A_320), NTD1-88 fused inteins
and Inteins respectively (see Fig. 14,15).

Figure 14: Size exclusion chromatogram of the ligation mixture after 1 hr of
incubation. UV absorption peaks are marked as peak1, peak2, peak 3, and peak 4.
16
Figure 15: 14% SDS-PAGE analysis of purification of the intein linked
Orb2A_1-320 purification. Lane 2 and 3 represents purification of
shorter construct of CTD89-320 fused intein in high- yield. Lane 5
represent the Orb2A_320 ligation reaction mixture in reaction buffer (4M
urea, 500mM NaCl, 0.1% tween, 50mM Tris, 2mM TCEP). Lane a and
b represent the purified ligated Orb2A_320. Lane c and d represent
NTD1-88 fused inteins and lane e represent Inteins.

3.5 Mass Spectrometry:
In order to characterize intein-linked Orb2A_320, we obtained intact protein analysis by
mass spectrometry. We acquired MALDI-TOF MS and electrospray ionization on the size-
exclusion fractions of intein-linked Orb2A_320 (concentration = 10µM). The predicted
mass of intein-linked Orb2A_320 (33.5 kDa) was in agreement with the MALDI-TOF MS
experimentally determined mass (see Fig. 16). We used the ExPASy - ProtParam tool to
predict the mass of intein-linked Orb2A_320. Since the concentration of the intein-linked
Orb2A_320 was low and the concentration of salt and urea was high in the buffer,
electrospray ionization signals were too low to deconvolute.
17

3.6 Expression and Purification of Non-ligated Orb2A_1- 320:
We expressed non-ligated Orb2A_320 in BL-21 (E.coli) strain to use it as a control for our
study. We purified them in the form of soluble proteins Ni-NTA chromatography followed
by size exclusion chromatography. We observed a strong peak at 260 nm after measuring
the sample concentration using UV-Vis spectroscopy (see Fig. 17). We measured a
260/280 ratio of 1.74 using the nanodrop spectrophotometer. The absorption at 260nm
suggested a nucleic acid contamination in the sample. To confirm the DNA contamination,
we ran an agarose gel on non-ligated Orb2A_320 and stained it with EtBr., however, we
observed no bands on the agarose gel (see Fig. 18). We postulated that perhaps the DNA
present in the sample was below the detection limit of EtBr and therefore decided to test
the sample with a more sensitive dye.
Figure 16: MALDI-TOF analysis of the intein-linked Orb2A_320. The protein was
purified by size exclusion chromatography (see Fig. lane a and b) and it was in
ligation reaction buffer (4M urea, 1 M NaCl, 50mM Tris, 2mM TCEP)
18
Figure 17: The UV absorption spectrum of the
Orb2A_320 size exclusion aliquots showed an
absorption peak at 260nm and 280nm.
exclusion size Orb2A_320
aliquots.
Figure 18: No bands were
observed in an agarose DNA gel
electrophoresis analysis of the

We used a fluorescent dye-based DNA assay to detect the DNA contamination. We used
the Qubit 4 fluorometer to measure the concentration of the DNA and RNA present in the
sample. The DNA and RNA concentrations were 28.6 ng/mL and 29.1 ng/mL, respectively
in non-ligated Orb2A_320. To remove nucleic acid contamination, we added DNase to
the lysis buffer and further purified the protein using a heparin column following the Ni-
NTA column. We observed a strong 280 nm peak in Orb2A_1-320 after the heparin
column (see Fig. 21).
19

We also observed an interesting event during the purification of OrbA_320, in which SDS-
page analysis showed the presence of two bands, 35 kDa and 30kDa in an almost 1:1
ratio after the heparin column. (see Fig. 20)

Peak 2
Peak 1
Figure 19: Heparin column chromatogram after Ni-NTA column. UV absorption
peaks marked peak 1 and peak2 were non-ligated Orb2A_320 and a protein
impurity, respectively.
Figure 21: The UV absorption spectrum of
the Orb2A_320 heparin column aliquots
displayed a prominent absorption peak at
280nm.
Figure 20: 14% SDS-
PAGE analysis displays
the Orb2A_320 double
band in the nickel and the
heparin purification
aliquots.
20
3.7 Intrinsically Disordered Orb2A_320 Protein Can Accumulate
in Droplet- like Assemblies

We eluted intein ligated Orb2A_320 and non-ligated Orb2A_320 from the size exclusion
column using a buffer with high ionic strength (i.e. 1M NaCl) to prevent droplet formation.
To investigate if the ligated and non-ligated Orb2A_320 constructs formed droplets, we
first concentrated these fractions using centrifugation filters to a working concentration of
at least 40 µM. To further probe the presence of droplets, we labeled the fractions of intein
ligated Orb2A_320 and non-ligated Orb2A_320 with the fluorescent dye using the PD-10
columns. We then used PD-10 columns to exchange the protein into droplet forming
conditions (10mM HEPES, 0.05%b-mercaptoethanol). After the exchange into this buffer,
we observed turbidity in the fractions eluted from the PD-10 columns. Furthermore, we
observed that the droplets formed by both ligated Orb2A_320 and non-ligated Orb2A_320
were spherical in shape and fused together into larger droplets (see Fig. 22).

3.8 Analysis of Amyloid Fibrils by Electron Microscopy

We measured TEM images of intein-linked Orb2A_320 and non-ligated Orb2A_320 to
validate the formation of amyloid fibrils. We applied the solution of intein linked
Orb2A_320 in droplet forming conditions after 48 hours and 72 hours. A gird for non-
ligated Orb2A_320 in droplet forming conditions was made after 52 hours. EM grids were
stained with uranyl acetate and visualized using a transmission electron microscope. We
observed some fibrils, but mostly non-specific aggregates for the intein-linked Orb2A_320
(see Fig. 23). After this, we postulated that perhaps tween, which is present in the ligation
reaction mixture, could be inhibiting fibril-formation. We moved forward with this hypothesis
Figure 22: Intein- ligated Orb2A_320 and non-ligated Orb2A_320 droplets observed
by fluorescent microscopy at room temperature.
21
and replaced tween with high salt (1M NaCl) in the reaction mixture.
For non-ligated Orb2A_320, we observed protofibrils along with some non-specific
aggregates (see Fig. 24).

Figure 24: TEM images of the non-linked Orb2A_320 after 52 hours.

Figure 23: TEM images of the intein-linked Orb2A_320 after 48 hours (upper
panel) and after 72 hours (lower panel).
22
3.9 Thioflavin T Assay:
We studied the aggregation kinetics of the Intein-ligated Orb2A_320 and non-ligated
Orb2A_320 by thioflavin t (Tht) fluorescence. We measured ThT fluorescence of the
following samples: 30 µM post-heparin non-ligated Orb2A_1-320 (concentrated),20 µM
post–heparin non-ligated Orb2a_1-320 (diluted) (see Fig. 25 ), and 10µM intein-linked
Orb2A_1-320 in the ligation reaction buffer (4 M urea, 1 M NaCl, 50 mM Tris, 2 mM TCEP)
(see Fig. 26). We observed that the ThT curve for intein inked Orb2A_320 had the
characteristic long lag phase, growth phase and final plateau commonly observed in Tht
kinetic assays for amyloid proteins (see Fig. 25,26).

Figure 25: Kinetics of aggregation of non-
ligated Orb2A monitored by ThT
fluorescence emission at different
23

Figure 26: Kinetics of aggregation of Intein-linked
Orb2A monitored by ThT fluorescence emission.
24
4. Discussions:
Here, we demonstrated an efficient method for the preparation of segmentally labeled
Orb2A achieved by protein trans-splicing reaction by Nostoc Punctiforme (Npu) DnaE
split intein. We observed robust trans-splicing with an efficiency of more than 90% in vitro
without any refolding procedures. Since trans-splicing requires cysteine at the+1position
of C-extein, we performed one cysteine mutation in the native amino acid sequence of
CTD89-320.

There are many studies that used naturally occurring intein mediated segmental isotope
labelling of domains for NMR structural studies (18,19,32). For example, split intein Ssp
DnaE has been used to perform trans-splicing in vivo under native conditions (18,19). Sara
Zuger and coworkers used Ssp DnaE to study proteins and their conformational changes
in living cells by in-cell NMR. They observed lower yield with intein-mediated protein
ligation compared with that of fusion protein prepared by conventional method (32). This
was presumably due to the lower in vivo reconstitution efficiency of Ssp DnaE. Another
naturally occurring Npu DnaEN is widely used because it is more tolerant of amino acid
substitutions (18,19). In general, superior trans-splicing activity was demonstrated by Npu
DnaE compared to Ssp DnaE. For example, Sebanti Gupta and coworkers obtained the
structure of HIV-1 tubular capsid protein assemblies by segmental labeling strategies
(18). Similarly, Tobias Schubeis and coworkers showed that the segmentally labelled
13
C
and
15
N-labeled prion domain of HET-s exhibits reduced spectral complexity in the solid-
state NMR spectra (19). They showed that a single Ser-to-Cys substitution introduced
between NTD and CTD segments didn’t perturb the structure of the protein (19).

We observed, yields of approximately 90% splicing product within 10 minutes, whereas
Tobias Schubeis and coworkers observed 30% yield of splicing product within 40 hours.
One possible explanation for the difference in ligation product yields and splicing rate is
the amino acid residues flanking inteins. In our case, +2 C-extein residue was tyrosine,
one of the most favorable residue types at this position while in Tobias and coworkers
HET-s construct, the +2 residue was aspartic acid, one of the most unfavorable residue
types at this position (19). Previous studies have shown that C-extein residues at the +2
residues of the intein affect the efficiency of the splicing reaction. In general, large
hydrophobic residues are preferred at the +2 position of the C-extein. For example,
mutating the native Phe+2 residue to Ala in the Npu extein sequence decreased the
splicing rate from 9 minutes to 50 minutes. Bulky +2 residue in extein helps in stabilizing
the last step of protein splicing, which involves the fast cyclization of Asn137. (33)
Together with other demonstrations (18,19) showed that adding His6 tag at the C-
terminus of the NTD-intein fusion protein and N-terminus of the CTD-intein fusion allowed
a single step separation. However, in our case a key complication arose due to the fact
that the intein-linked Orb2A also binds the Ni-NTA column because it has a histidine rich
domain in its sequence. We utilized different purification strategies such as dialysis, ion
exchange chromatography, and size exclusion chromatography however, none of them
were able to separate intein-linked orb2A from the educts; the NTD1-88 fused intein and
the inteins were eluted together in all the purification techniques mentioned above. One
25
possible explanation for this problem is that the ligation product is interacting non-
specifically with the educts of the trans-splicing reaction. This argument was supported
by the fact that including tween 20 in the reaction mixture helped getting better separation
of the intein-linked product and the educts by size-exclusion chromatography.

In the future, we are planning to label the N-terminal domain of Orb2A with
15
N and
13
C
isotopes first by growing NTD1-88 fused intein in the isotopically enriched medium and
performing in-vitro trans-splicing with unlabeled CTD89-320 fused intein. This will allow
us to generate segmentally labelled full-length Orb2A with a simplified NMR spectra. We
also plan to use a similar labelling strategy for Orb2B. In segmentally labelled Orb2B, we
are planning to replace the His6 tag with strep tag to achieve single step purification of
the reaction product. We are hoping that these results will help us answer twoquestions:
(i) location of amyloid core of Orb2A? (ii) why Orb2A can aggregate more quickly than
Orb2B. Overall, we hope that this knowledge will bring us closer to understanding how
Orb2 aggregation regulates the translation of the target mRNA, thereby revealing the
molecular mechanism of long-term memory formation.
26
5. Materials and Methods:

5.1 Plasmid Design

We chose to use split intein DnaE from Nostoc Punctiforme because it can tolerate many
different amino acids at the splicing junction and remains active under a wide range of
conditions, including a high concentration of denaturing agents.
DNA sequences containing NTD1-88 fused intein, CTD89-540 fused intein and the
CTD89-320 fused intein constructs were synthesized by GenScript (GenScript USA Inc.).
As shown in the figure (27) the NTD1-88 fused intein fusion sequence contains 1-88
amino acids residues of Orb2A along with the N- terminal intein segment of Npu DnaE.
We used pET28 vector, which contains the T7 promoter and a kanamycin resistance
gene. The DNA sequence was cloned using the restriction sites Ncol and Xhol, thereby
obtaining a final DNA sequence containing a His tag at the C-terminal domain as well as
two extra amino acids, both of which were removed later with the rest of the intein. We
specified Tyrosine substitution in position two with Glycine, because Ncol recognizes (5’
C* CATGG 3’). We used two different CTD89-540 fused intein and CTD89-320 fused
intein sequences. The first contained amino acid residues 89-540 of Orb2A and a shorter
construct residues 89-320 of Orb2A along with C-terminal intein segment of Npu DnaE.
In this case, we used pET11a vector containing the T7 promoter and an ampicillin
resistance gene. The DNA sequence was cloned using the restriction sites Ndel and
BamHI. To make the shorter Orb2A CTD89-320 fused intein construct (1-320), we simply
added a stop codon after 320 amino acids. The CTD89-540 fused intein and CTD89-320
fused intein sequences contained a His6tag at the N-terminus. All these constructs were
codon optimized for expression in E.coli . Vectors were checked for accuracy by
transforming into competent XL10-Gold cells. Plates were incubated overnight at 37°C
and a single colony was inoculated in the LB Miller Broth for 18 – 20 hours at 37°C and
225 rpm. Zyppy palsmid miniprep kit was used to isolate DNA from overnight cultures.
Concentration of DNA was obtained using Nanodrop ND – 1000 spectrophotometer and
sent out for sequencing.
27

Figure 27: Schematic representation of the plasmid design and the purification strategy
for the preparation of the segmentally labeled Orb2A.

5.2 Transformation and Protein Expression:
5.2.1 Transformation:
Competent BL21 (DE3) cell aliquots (200 µL) were taken from -80°C refrigerator and
allowed to thaw on ice. DNA (2ng) was added to the pre-cooled culture tube containing
cells. The cell DNA mixture was incubated on ice for 10 minutes and transformed using
heat shock in a water bath at 42°C for 30 seconds. The culture tube was put back on ice
for 2 min and then 800 µL of SOC medium (without antibiotics) was added to the cell DNA
mixture. The culture tubes were then incubated at 37°C and 225 rpm (series 25 Incubator
Shaker) for less than an hour. 50-200ul of the mixture were plated on a 10 cm LB agar
plate containing the appropriate antibiotics. Plates were incubated overnight at 37°C.

5.2.2 Protein Expression:
A single colony of transformed BL21(DE3) was picked from the plates and used to
inoculate 25 mL of LB Miller Broth containing appropriate antibiotics. For Orb2A NTD1-
88 fused intein, cell cultures were incubated at 30°C and 200 rpm for 15 – 18 hours
(Innova™ 4400 Incubator Shaker). For Orb2A CTD89-540 fused intein shorter and
CTD89-320 fused intein, cell cultures were incubated at 37 °C and 225 rpm for 3-4 hours.
The culture was expanded by adding an aliquot (3mL) of the starter culture into 500mL of
LB Broth with 500 µL of appropriate antibiotics. The culture was then incubated at 37°C
and 225 rpm until the culture density of OD 600 reached 0.6. The expression was induced
by adding 500 µL Isopropyl 1-thiol-β-D-galactopyranoside (1M). Cell cultures were then
28
switched to 25°C for NTD1-88 fused intein and 16°C for CTD89-540 fused intein with
shaking at 160 rpm and incubated for 15 – 18 hours. To collect cell pellets, cells were
centrifuged at 4,000 rpm for 20 min using a Sorvall SLC-6000 rotor. The Supernatant was
removed, and cell pellets were stored at -80°C.

5.3 Cell Lysis
5.3.1 For Orb2A NTD1-88 fused intein Segment:
Cell pellets were taken out from -80°C and allowed to thaw on ice for 15 minutes and
resuspended in 25 mL denaturing buffer (100 mM NaH2PO4, pH 8.00, 10 mM Tris, 8 M
urea and 0.05% (v/v) β –mercaptoethanol). Cells were vortexed until they were
completely dissolved. Cells were lysed using Cell Disruptor Sonicator (Heat System
Model W-220F) set at amp 75 for 6 minutes (with 30 secs pause after 1 min). The cell
lysate was spun at 20,000 rpm for 20 min at 4°C using Sorvall ss-34 rotor and the
Sorvall
®
Evolution RC centrifuge to pellet the cellular debris. After centrifugation, the
supernatant (cleared lysate) was saved for the Ni-NTA purification.

5.3.2 For Orb2A CTD89-540 Fused Intein Segment (Inclusion Body
Purification):
Cell pellets were taken out from -80°C and allowed to thaw on ice for 15 minutes and
resuspended in 20mL lysis buffer (50mM Tris, 100mM NaCl, 0.5%TritonX, 0.05% (v/v) β
– mercaptoethanol, 0.1mg/ml lysozyme). The cells were lysed using Cell Disruptor
Sonicator (Heat System Model W-220F) set at amp 80% for 9 minutes (with 30 secs pause
after 1 min). The cell lysate was centrifuged at 10,000 rpm for 15 minutes at 4°C using
Sorvall ss-34 rotor and the supernatant was discarded. Inclusion body pellets were
washed with 20 mL lysis buffer, which was sonicated and centrifuged again as above.
Inclusion Bodies were resuspended in denaturing buffer (6 M Gnd-HCl, 100 mM
NaH2PO4, 250 mM NaCl, 10% glycerol, and 0.05% (v/v) β – mercaptoethanol) and broken
into smaller chunks by vortexing. The solution was then sonicated as above and left over-
night at room temperature to solubilize inclusion bodies. The solubilized inclusion bodies
were spun at 20,000 rpm for 30 minutes at 4°C. After centrifugation supernatant (cleared
lysate) was saved for the Ni-NTA purification.
29
5.3.3 Cell Lysis of Non-ligated Orb2A_320 (DNase Treatment):
Cell pellets were taken out from -80°C and allowed to thaw on ice for 15 minutes,
resuspended in 100 mL lysis buffer (1 M urea, 100 Mm KCl, 10 mM HEPES, 0.05% (v/v)
β – mercaptoethanol,10 µL DNase, 75 Mm Imidazole, and 5 mM MgCl2) and left on ice
for 10 minutes. The cells were lysed using Cell Disruptor Sonicator (Heat System Model
W-220F) set at amp 100% for 9 minutes (with 30 secs pause after 1 min). The cell lysate
was spun at 20,000 rpm for 20 min at 4°C using Sorvall ss-34 rotor and the the
Sorvall
®
Evolution RC centrifuge to pellet the cellular debris. After centrifugation, the
supernatant (cleared lysate) was saved for the Ni-NTA purification.

5.4 Purification:
Buffer Used for NTD1-88 fused intein Purification:

Denaturing Buffer (pH 8.0)
100 mM NaH2PO4
10 mM Tris
8 M urea
0.05% (v/v) β – mercaptoethanol

Renaturing Buffer (pH 8.0)
50 mM NaH2PO4
200 Mm NaCl
10% glycerol
0.05% (v/v) β – mercaptoethanol

Buffer Used for CTD89-540 Fused Intein Purification:
Denaturing Buffer (pH 8.0)
100 mM NaH2PO4
10% glycerol
8M urea
0.05% (v/v) β – mercaptoethanol

Elution Buffer (pH 3.75):
100 mM NaH2PO4
50% glycerol
8M urea
250 mM NaCl
10 mM citric acid
0.05% (v/v) β – mercaptoethanol
30
For the Ni-NTA gravity column, 10 mL of Ni-NTA slurry (Sigma- Aldrich) was added and
it was equilibrated with 25 mL of denaturing buffer (no β – mercaptoethanol). The soluble
portion (supernatant) of the cell lysate was poured onto the pre-equilibrated IMAC column
after which the column was left on a shaker and incubated on the shaker at room
temperature for 1–1.5 hours. Flow-through was collected and column was washed with
25 mL of denaturing buffer containing 0.5% Triton X-100. The resin was further washed
by 25 mL denaturing buffer containing 500 mM NaCl, and 25 mL denaturing buffer pH
6.75. NTD1-88 fused intein was eluted from the column using an Imidazole step gradient
in renaturing buffer. The imidazole gradient steps were 100 mM, 150 mM, 200 mM, and
250 mM 10 mL each.

CTD89-540 fused intein was eluted from the column using the 6 mL of elution buffer
three times. The maximum concentration of CTD89-540 fused intein was generally eluted
in the second fraction.

An aliquot (40 µL) of whole cell lysate, pellet, supernatant, flow through, washes and
elutions were collected for analysis by SDS-PAGE. Column elutions were shock frozen
using liquid nitrogen and stored at -80°C.

Purification of the Non-ligated Orb2A_320 Using Heparin Column:

A heparin column was used to further purify the non-ligated orb2A_320 after the initial Ni-
NTA column using the Fast Protein Liquid Chromatography (FPLC) system. A HiTrap
Heparin HP column (GE healthcare) was used for the purification of non-ligated
Orb2A_320. The column was equilibrated with 5 column volumes of deionized water and
5 column volumes of binding buffer (1 M urea, 10 mM NaH2PO4, and 0.05% (v/v) β-
mercaptoethanol pH 7.0). For sample preparation, 5 mL of the nickel column elute was
diluted 30 times to lower the ionic strength and promote interactions between the sample
and the resin. Sample (150 mL) with the flow rate of 1.5 ml/min was loaded onto the
column, which was followed by 5 CV wash by binding buffer. It was eluted with 2 column
volume of binding buffer and using continuous 2 M NaCl salt gradient. Column elutions
were shock frozen using liquid nitrogen and stored at -80°C.

5.4.1 Intein-linked Orb2A Preparation:

In order to ligate the N and C-terminus of Orb2A, samples were buffer exchanged into 4
M urea, 50 mM Tris, 150 mM NaCl, and 1.0 mM TCEP at pH 7.2 using the PD- 10
desalting columns (GE healthcare). The trans-splicing reaction buffer of the intein-linked
Orb2A (1-320) contained 4 M urea, 50 mM Tris, 500 mM NaCl, 0.1% tween and 1.0 mM
TCEP at pH 7.2. After exchanging the buffer, protein concentrations were measured by
UV absorption at 280 nm. Because NTD1-88 fused intein was eluted in high-yield it was
used in excess for the trans-splicing reaction so that CTD89-540 fused intein was all used
up during the reaction. The 2:1 mixture of the eluted NTD1-88 fused intein and CTD89-
540 fused intein proteins were incubated at the room temperature for 24 hours and the
CTD89-320 fused intein was incubated for 1 hour. Samples for the SDS-PAGE analysis
were taken at several time points to follow the trans-splicing reaction.
31
5.5 Troubleshooting Trans-splicing Reaction Product
Purification:
5.5.1 Purification of the Reaction Product Using Ni-NTA column:
Ni-NTA slurry (5mL) (Sigma- Aldrich) was added to a gravity column and equilibrated the
column with denaturing buffer (4 M urea, 10% glycerol, 10 mM NaH2PO4, and 0.05% (v/v)
β – mercaptoethanol). 5 mL of the ligation reaction mixture was added to the column and
incubated at room temperature for 1-1.5 hours. Flow-through was collected and the
column was washed with 10 mL denaturing buffer containing 0.5% Triton X-100. It was
further washed with 10 mL denaturing buffer containing 500 mM NaCl. An imidazole step
gradient, for example, 20, 50, 100, 150, 200, 250 mM imidazole concentration was used
to elute the protein. Aliquots (40 µL) of flow-through, washes as well as elutions were
collected for analysis by SDS – PAGE.

5.5.2 Purification of the Reaction Product Using Dialysis:
Dialysis Tubing with the molecular weight cut off 30 kDa (Spectra/Por
®
Molecularporous
Membrane Tubing) was placed in the 1 L buffer containing 4 M urea, 50 mM Tris, 150
mM NaCl and 0.05% (v/v) β – mercaptoethanol at pH 7.2. The Sample was allowed to
dialyze at room temperature for 2 hours. The buffer was then changed, and the sample
was dialyzed for another 2 hours. After this, buffer was changed again and left overnight
at room temperature.

5.5.3 Purification of the Reaction Product Using Ion Exchange
Chromatography:
HiTrap Q HP ion exchange chromatography column (GE Healthcare Life Sciences) was
used. A cation exchange resin was charged at pH 6.5, so that the negatively charged
ligation product (PI > 7.5) was bound to a positively charged resin. To charge resin in the
pH range needed, 5 CV of 200 mM Sodium Acetate (pH 4.5) followed by 50 mM Sodium
Acetate (pH 4.5) was added to the column. Finally, 5 CV of equilibration buffer (4 M urea,
50 mM Tris, and 0.05% (v/v) β – mercaptoethanol pH 6.5) was added to the column. For
sample preparation, the intein reaction mixture was diluted 15 times with 10 Mm Tris pH
6.5. Sample was loaded onto a pre-equilibrated cation exchange chromatography column
and left on a shaker for 1-1.5 hours at room-temperature. The column was washed with
the buffer containing 4 M urea, 50 mM Tris, 200 mM NaCl, and 0.05% (v/v) β –
mercaptoethanol pH 6.5. A continuous salt gradient was used to elute the ligation product.
The samples were eluted from the column using 3 CV elution buffer (2 mL fractions) which
contained 4 M urea, 50 mM Tris, and 0.05% (v/v) β – mercaptoethanol at pH 6.5. To
charge the resin at pH 6.0, pH 7.0, and pH 7.5, same procedure was followed by adjusting
the pH of the equilibration buffers accordingly.
32
5.5.4 Purification of the Product Using Size Exclusion
Chromatography:
The size exclusion chromatography was carried out using the FPLC system (modular
NGC chromatography system) and utilizing the pre- packed columns from GE Healthcare
(HiPrep 16/60 Sephacryl S-300 HR column, 16 mm × 600 mm and HiPrep 16/60
Sephacryl S-200 HR column, 16 mm × 600 mm). Buffers and the sample were passed
through a 0.22µM filter membrane. Further, buffers were degassed before loading it onto
the size exclusion column. The column was equilibrated with the 2 CV deionized water
followed by 2 CV ligation reaction buffer (4 M urea, 1 M NaCl, 50 mM Tris, 0.05% (v/v) β
– mercaptoethanol at pH 7.2). The flow rate of 0.5 (mL/min) was selected to maximize
resolution and minimize the separation time. The ligation reaction mixture was loaded
onto the column. The ligation product was eluted using 1 CV elution buffer collected in
the 2 mL fractions. The column was cleaned with 1 CV cleaning buffer containing 6 M
GuHCl, 250 mM NaCl, 100 mM NaH2PO4 0.05% (v/v) β – mercaptoethanol pH 8.0
followed by 2 CV deionized water and 2 CV 20% ethanol.

5.6 UV spectroscopy:
5.6.1 UV 280 nm:
Absorbance at 280nm was determined using a spectrophotometer (Cary 60 UV – Vis,
Agilent Technologies) and equation A = εcl. The spectrophotometer was set to collect
spectra over a wavelength range of 250 – 350 nm and using quartz glass cuvettes (10mm
light path). The baseline was taken using 500 µL of protein elution buffer.

5.6.2 Fluorescamine Assay:
Fluorescamine reagent was made using 5 mg fluorescamine in 10 ml Acetone. Borate
buffer (0.1M Borate, 1% SDS pH 9.0) was used as a diluting reagent to make the standard
curve using lysozyme. Lysozyme was prepared at 1mg/ml. The concentration was verified
using a Cary 60 UV Spectrophotometer (Cary 60 UV – Vis, Agilent Technologies). The
reagents were combined in the following manner.

Tube Borate Buffer Sample buffer Protein
Blank 1 ml
100 µl 0 µl lysozyme
1 1 ml
90 µl 10 µl lysozyme
2 1 ml 80 µl 20 µl lysozyme
3 1 ml
70 µl 30 µl lysozyme
4 1 ml
60 µl 40 µl lysozyme
33
5 1 ml
50 µl 50 µl lysozyme
Sample no dilution 1 ml
0 µl 100 µl sample
Sample 1:1 dilution 1 ml
50 µl 50 µl sample

Each sample was boiled at 95ºC for 5 min on a hot plate and was allowed to cool down
to room temperature. 125 µL fluorescamine was added to each tube dropwise (using a
glass syringe) followed by vortexing immediately after a couple of drops. 200 µL of the
sample from each tube was added to 96-well plate. Fluorimeter (Eppendorf Plate Reader
AF2200) with excitation at 450nm and emission at 484 nm was used to measure the
fluorescence. A standard curve was plotted using the lysozyme concentration from the
UV scan and the regression line was fitted to data. The resulting equation was used to
calculate the concentration of the samples.

5.7 Formation of Droplets and Amyloid Fibrils Assemblies:
Purified, intein-linked Orb2A_320 was concentrated to 40 µM in the trans-splicing reaction
buffer (4M urea, 50mM Tris, 500mM NaCl, 0.1% Tween and 1.0 mM TCEP pH 7.2), using
centrifugal filter devices (Amicon Ultra, 6ml volume, 3kDa cutoff). The filter device was
rinsed with the ligation reaction buffer for 10 mins 4000 rpm at 4°C prior to use to remove
any trace amount of glycerine on the membrane. To increase the concentration of the
intein-linked Orb2A_320, intein-linked Orb2A_320 (2 mL) was added to the device and
centrifuged at 4,000 rpm for approximately 40 minutes at 4°C using a swinging bucket
rotor (Eppendorf Centrifuge 5804 R). The concentrated sample was recovered from the
centrifuge tube by inserting the pipette into the bottom of the filter-device and using the
side-to-side sweeping motion to withdraw the sample. To trigger a droplet assembly, the
reaction buffer of the concentrated intein-linked Orb2A was desalted into buffer containing
10 mM HEPES, 5 mM TCEP. PD MidiTrap G-25 (GE healthcare) was used for desalting.
Th eluted fraction of the protein was turbid, and it was incubated at room temperaturefor
48 hours to promote the fibril formation.

5.8 Transmission Electron Microscopy
TEM images were recorded with a JEOL 1400 transmission electron microscope
operated at 100 keV. Negatively stained samples were prepared on a Carbon-coated
formvar mounted on the copper grid. 10µL of the intein-linked Orb2A_320 droplet
assembly solution at different time points, such as 0h, 12h, 24h, 48h and 72h, were
absorbed to the carbon film for 5 mins. Excess sample was removed from the grids using
filter paper. It was stained with 1% uranyl acetate for 2 mins, rinsed with 1% uranyl acetate
and deionized water respectively, and dried in air.

5.9 Thioflavin T staining
The kinetics of amyloid fibril formation were studied using Thioflavin T (Tht) fluorescence
34
at 484 nm (Eppendorf Plate Reader AF2200). Samples (100uL) were incubated with 2µL
of Tht stock solution, which is prepared at 5mM in deionized H2O and thawed for 30
minutes before use. A similar baseline solution was made consisting of 100 µL of the
buffer (10mM HEPES, 5mM TCEP) incubated with the 2µL Tht stock solution.
Fluorescence was measured with excitation at 450nm and emission at 484 nm was used
for measuring the parameters.
35
6. References:
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Abstract (if available)

Abstract Solid-state NMR has become a powerful tool for studying the structure, dynamics and interaction of supramolecular protein assemblies under near physiological conditions. The major limitation of protein NMR is its spectral crowding resulting in resonance overlap, which increases with the number of residues. Segmental isotope labeling allows specific segments within the protein to be identified by NMR and thus reduces the spectral complexity. In my thesis, I will present my work on the split intein DnaE from Nostoc punctiforme to apply sparse isotope labeling strategies to the functional amyloid Orb2A. Split-inteins are protein introns that splice out autocatalytically joining the target protein by peptide bond. We presented an efficient method for production of the segmentally labeled Orb2A in which either the N or C terminal domain is uniformly labelled with ¹⁵N, ¹³C. The NTD of Orb2A was fused with the NTD of Npu DnaE (NTD1-88 fused intein) and its CTD was fused with the CTD of Npu DnaE (CTD88-540 fused intein). We observed high-yield the intein fusion proteins. Additionally, robust trans-splicing with an efficiency of more than 90% splicing product was obtained. However, we observed difficulty in separating ligation products from the educts because the original idea of separating ligation product from Ni-NTA column failed due to the presence of histidine domain in the intein-linked Orb2A. We utilized different purification strategies such as dialysis, ion exchange chromatography, and size exclusion chromatography, however, none of them were able to separate intein-linked orb2A from the educts

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Creator Garg, Samridhi (author)

Core Title Facilitating unambiguous NMR assignment by solid–state NMR using segmental isotope labeling through split-inteins

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 07/28/2019

Defense Date 05/29/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag functional amyloids,inteins,long term memory formation,OAI-PMH Harvest,protein purification,solid-state NMR

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Language English

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Advisor Siemer, Ansgar (committee chair), Camarero, Julio (committee member), Langen, Ralf (committee member)

Creator Email samridhi.garg100@gmail.com,samridhl@usc.edu

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