Summer Research Fellowship Programme of India's Science Academies 2017
Purification and solution state conformation of recombinant
human gamma C crystallin and studying the quality of donor
cornea
Abhinav Pokhriyal
Sri Venkateswara College, New Delhi
Guided by
D. Balasubramanian
L.V. Prasad Eye Institute, Banjara Hills, Hyderabad 500034
1. Introduction
The lens helps focus light rays passing through it and on to the retina. It is unique in the sense that
with age, cells of the lens are not turned over or lost as a consequence of which it both grows in
size and weight with age. The major constituent of eye lens is water, nearly 2/3rd, the rest is
protein. Such a large protein content is responsible for the high refractive index of the lens. Lens
is essentially fiber cells surrounded by a single layer of cuboidal epithelial cells on the anterior
surface. The whole structure is surrounded by a capsule. Lens fiber cells are formed because of
differentiation of epithelial cells where they lose light scattering organelles like nucleus,
mitochondria etc. Lens can be divided into several regions, the nucleus which has cells of
embryonic and fetal origin which is surrounded by cortex where new cells are added in the cortical
and the epithelial region.
Transparency of the lens is required so that light can pass through it and form a clear image on the
retina. This transparency of lens is imparted by loss of organelles, no vascular system and primarily
the presence of proteins known as crystallins.
Crystallins form the bulk of lens proteins (90%). Two major types of crystallins are α and βγ
crystallins. All these proteins are water soluble.
α crystallins belong to the family of heat shock proteins that exhibit a chaperone like activity. They
are multimeric proteins that account for nearly 30% of total crystallins. As lens proteins denature
under stress α crystallins act on them so that these proteins can be refolded. Absence or decreased
efficiency of this system results in aggregate formation that scatters light and can hamper normal
vision. They are of two types of subunits αA and αB. They have the highest electrophoretic
mobility towards anode.
β crystallins are multimeric proteins that account for 30% of all crystallins. There are four acidic
A1, A2, A3 and A4) and three basic (β B1, B2 and B3) β crystallins. γ family has three members,
γ C, D and S. They constitute 25% of all crystallins and are monomeric proteins. They are
synthesized when epithelial cells start to differentiate to form fiber cells. They also have two
similar domains with each domain having 2 super secondary structure elements calls Greek Key
motif. Mutations in Greek key motif makes the protein sparingly soluble and self-aggregate, the
protein becomes less stable and aggregate to scatter light.
1.1. γ-crystallins
γ crystallins are monomeric proteins having a molecular weight of about 21 kd. Mammals are
known to contain seven γ crystallin genes of which six genes gamma AF are closely linked having
high sequence similarity. 7th gene, γS crystallin is located on a different chromosome and is
divergent from the others. It is expressed in non-ocular tissues also. In human lens only γC and γD
crystallins are expressed abundantly whereas γE and γF crystallins are pseudogenes. Data from
protein sequence and x ray crystallography shows that they are structurally related and belong to
a single βγ protein superfamily. γ crystallins are made up of 2 similar domains which have 2 similar
structural motifs. Motifs 1 and 2 are located on the N terminal whereas motifs 3 and 4 are located
on c terminal. These are consecutive along the polypeptide chain with a single interdomain
connecting peptide.
βγ crystallin has a characteristic domain which is made of 2 consecutive greek key motifs, each
having strands that intercalate to form sheets that pack together to form a β sandwich domain.
The 2 domains have a complex topology where each linear 2 stranded motifs exchange its 3rd β
strand to a β sheet belonging to a partner motif.
However, the difference between β and γ crystallin groups are
Motifs of β crystallin are coded by separate exons whereas those of γ crystallin are coded
by a single exon.
β crystallins have long N and C terminal sequence extensions.
Long linker of β crystallin is straight and allows intermolecular quaternary interactions
between 2 β crystallins. On the other hand, the V shaped linker of γ crystallin allows for
intramolecular domain interaction.
Fig. 1. Shows how the greek key motifs one in the N-terminal segment (blue) and the other in the
C- terminal (red) are folded into making a compact globular structure.
2. Techniques used
2.1. Sonication
Source taken from: Principles and techniques of Biochemistry and Molecular Biology (7th
edition) by Keith Wilson and John Walker.
A sonicator probe is lowered into the suspension of cultured cells and high frequency sound waves
(> 20 kHz) generated for a short duration that causes disruption of cells by shear force and
cavitation. Cavitation are areas undergoing alternate compression and rarefaction, that rapidly
interchange. The gas bubbles in the buffer are initially under pressure but, as they decompress,
shock waves are released which disrupt the cells. Only small samples (50100 cc) can be handled.
As heat is produced during sonication, it becomes imperative that samples be kept on ice during
treatment.
(NH
4
)
2
SO
4
precipitation: Upon the addition of salt (<0.15 M) the solubility of proteins increases,
an effect termed salting-in. However, at higher salt concentrations, protein solubility decreases and
thus they precipitate; this effect is termed salting-out.
2.2. Hydrophobic interaction chromatography
Source taken from: Principles and techniques of Biochemistry and Molecular Biology (7th
edition) by Keith Wilson and John Walker.
This type of chromatography exploits the surface hydrophobicity of proteins. In pure water, the
water molecules cover the hydrophilic regions of the proteins thus masking the hydrophobic
groups located in the core of the protein. However, upon addition of salt, water is used to solvate
the salt molecules as a consequence of which hydrophobic groups on the proteins are exposed
which can interact with hydrophobic groups (alkyl or aryl) coupled to the matrix. Commercially
available matrices include Phenyl Sepharose, Phenyl SPW etc. A porous matrix is used to provide
high internal surface area which is equilibrated with the buffer having the same salt concentration
as in our sample. The type of salt and its concentration in the start buffer depends on whether the
proteins of interest bind to the column and majority of contaminating proteins pass directly through
the column. Proteins are eluted by decreasing the salt concentration in the elution buffer. As the
level of salt decreases proteins with the lower hydrophobicity are the first to be eluted from the
column. Proteins can thus be eluted differentially in a purified, concentrated form. Proteins with
the highest hydrophobicity will be most strongly retained and will be eluted last. The salt-free
buffer helps remove most tightly bound proteins at the end of an elution.
Gel filtration chromatography: It is also known as size exclusion chromatography and involves
the separation of molecules based on their size. Matrix here consists of porous beads which can be
made of silica, polyvinyl acetate, cross linked dextran etc. Pore size to be used depends on the size
of the protein to be eluted. Analytes that cannot pass through the pore will pass through the
interstitial space between the beads and will appear first in the eluate. Analytes that enter the beads
will pass through the column at a slower rate and hence appear in the later stages of the elution.
Gel filtration chromatography can be used for a variety of purposes like protein purification,
relative molecular mass determination and desalting.
2.3. Fluorescence spectroscopy
Source taken from: Fluorescence spectrophotometry by Peter TC So (MIT, USA) and Chen Y
Dong (MIT, USA).
When an electron in an excited molecule returns to its ground state, the corresponding energy is
emitted as radiation. This is known as fluorescence. As the electron is excited to a higher energy
level it loses some of its energy by collisions to reach the vibrational ground state of the excited
state. This is non-radiative loss and is known as internal conversion. Thus, the excitation energy
is greater than the emission energy hence the excitation wavelength is lower as compared to
emission wavelength.
Fluorescence spectrophotometry analyses fluorescence from a sample. Typically, UV light is used
to excite the electrons of the analyte. Fluorimeters are used to measure fluorescence. The
measurement of fluorescence signals is useful for monitoring the biochemical environment of a
fluorophore. Fluorescence intensity measurement helps determine the presence of fluorophores
and their concentrations. A typical fluorimeter has a light source, a specimen chamber with
integrated optical components, and high sensitivity detectors. Xenon arc lamps can be used as a
light source. The optical paths of the excitation and the detection light are along the orthogonal
axis thus ensuring minimal leakage of excitation light into the detection side. High sensitivity
photodetectors such as photomultipliers or charge-coupled device cameras are commonly used.
Monochromators or band-pass filters are placed along the paths of excitation and emission light to
select a specific spectral band. To measure the excitation spectrum the fluorescent intensity is
measured as a function of excitation wavelength at a constant emission wavelength whereas to
measure the emission spectrum the fluorescent intensity is measured as a function of emission
wavelength at a constant excitation wavelength.
Circular dichroism spectroscopy: It is a spectroscopic technique that measures the circular
dichroism of a molecule over a range of wavelengths. Circular dichroism is defined as the
[
]
= 100
ln 10
4
180
= 3298.2
difference between the absorption of left and right circularly polarized light by a chiral molecule.
A circularly polarized light is produced by the resultant of E vectors of 2 electromagnetic waves
that are ¼ wavelength out of phase and perpendicular to each other. Molar ellipticity is used to
display graphs of CD spectra.
The units of molar ellipticity are deg·cm
2
/dmol. Circular dichroism spectroscopy helps
characterize the secondary structure of a protein and how it is affected by changing the pH or the
temperature or in the presence of denaturants. It also helps compare the structures of proteins
obtained from different sources or comparing structures of different mutants of the same protein.
It can be used to determine how the protein’s secondary structure changes upon interaction with
other proteins or ligands.
An electro-optic modulator which is a crystal, transmits either the left- or right-handed circularly
polarized light through the sample, depending on the polarity of the electric field that is applied by
alternating currents. The photomultiplier detector produces a voltage proportional to the ellipticity
of the resultant beam emerging from the sample. The light source of the spectrometer is
continuously flushed with nitrogen to avoid the formation of ozone and help to maintain the lamp.
3. Methodology
For the project, E. coli cells expressing a recombinant human gamma C crystallin were used. The
protein is 174 amino acid long. The protein is a monomer with a molecular weight of nearly 21
kDa. This protein is abundantly expressed in human eye lens and several mutations in the gene
encoding this protein have been associated with cataract. The specific mutant used for our
experiments was R48H (Arg to His at codon 48) which has been reported to be associated with
congenital cataract.
For cell lysis and further downstream processing 1 litre, 50 mM Tris HCl buffer of pH 7.3 was
prepared by by dissolving 6.057 g of Tris Buffer in 996.27 ml of Milli-Q. 3.73 ml of concentrated
HCl (36%, 11.65M) was added to it. The pH was cross checked with a pH meter.
20 ml of lysis buffer was prepared. The components of the lysis buffer, their concentrations and
volumes are as follows:
Component
Stock concentration
Working concentration
Volume (ml)
Tris HCl buffer
5 0mM
46.3 mM
18.52
KCl
2 M
100 mM
1
EDTA
0.5 M
1 mM
0.04
DTT
0.1 M
1 mM
0.2
PMSF
100 mM
1 mM
0.2
Aprotinin
10 mg/ml
20 µg/ml
0.04
Recombinant protein expressing E. coli cell pellet available in the lab was suspended in 20 ml lysis
buffer. KCl is used to create an osmotic gradient to cause cell lysis. Ethylene Diamine Tetra
Acetate (EDTA) chelates metal ions thus the metal ions in the cell lines will not interact with the
protein and thus protein will remain in its native state. Moreover, chelation of metal ions by EDTA
ensures that proteases requiring metal ions as cofactors will remain inactive thus we will obtain
the intact protein. Dithiothreitol (DTT) is a reducing agent that keeps sulfide groups of the protein
in SH form thus ensuring that no intra or inter disulfide bridge formation takes place. Therefore,
the protein we obtain will be monomeric. Both phenylmethylsulfonyl fluoride (PMSF) and
aprotinin are protease inhibitors.
The pellet was dissolved in lysis buffer. The solution was sonicated for 40 cycles (5 s bursts of
sonication followed by 10 s rest cycle) at 34% amplitude at 4°C using a high intensity Ultrasonic
processor (Sonics Vibra Cell; Sonics & Materials Inc, Newton, MA). The cell lysate was
centrifuged at 30,000g, 4°C for 22 min using SORVALL RC 5C PLUS centrifuge. The supernatant
and pellet were separated. Nearly 15 ml of supernatant was obtained that had both the recombinant
protein and the protein of the host cell.
The supernatant was then subjected to (NH
4
)
2
SO
4
precipitation at 30% concentration. For this 2.64
g of (NH
4
)
2
SO
4
was added pinch wise to the solution kept on ice on a magnetic stirrer. Following
the addition of salt the solution was incubated on ice for 30 min. The solution was then suspended
at 30,000 g, 4°C for 22 min using SORVALL RC 5C PLUS centrifuge. The supernatant and pellet
were separated.
Supernatant was then used to perform Hydrophobic Interaction Chromatography (HIC). For HIC
a phenyl sepharose column was used. Phenyl sepharose is made using agarose beads with phenyl
groups attached via uncharged, chemically stable ether linkages. Phenyl being a hydrophobic
group binds to hydrophobic groups on the proteins to various extent. The matrix is rigid,
microporous with good capacity and low non-specific binding. Prior to passing the sample the
column was first equilibrated using 50 ml Tris HCl buffer having 30% (NH
4
)
2
SO
4
(8.6 g). This is
done to ensure that the buffer and the sample are in the same condition.
Sample was then loaded on the column. For elution, a linear decreasing gradient was used. The
decreasing gradient was prepared solution mixer having 2 chambers. The chamber with the outlet
for the solution to move into the column was filled with Tris HCl buffer having 30% (NH
4
)
2
SO
4
.
The other chamber was filled with the same volume of pure Tris HCl buffer. The former column
was fitted with a rotor to ensure that the solution is mixed uniformly prior to moving out. Total 11
fractions of 2.5 ml were collected.
Fractions 58 were pooled. The pooled sample was then concentrated by ultrafiltration using
amicon ultra centrifugal filters having a cut off membrane of 3 kDa. The sample was then
centrifuged at 4°C, 3500 rpm for 47min (Eppendorf centrifuge 5810R). Approximately 3 ml of
concentrate was obtained.
The concentrate was then used to perform gel filtration chromatography. For gel filtration
chromatography G-75 Sephadex was used. Sephadex is a trademark for beaded gel filtration
medium cross linked with epichlorohydrin under alkaline conditions. Molecules with a molecular
weight <80,000 Da are able to enter the porous beads. The column was equilibrated with Tris HCl
buffer of pH 7.3. The sample was then loaded on the gel filtration column and then the same buffer
was used to elute out the proteins. The void volume of the column was discarded (nearly 50 ml of
the initial eluent). Sample collection was then initiated and 40 fractions each of 2.5 ml were
collected.
Aliquots of crude pellet, supernatant and pellet of (NH
4
)
2
SO
4
fractionation, fractions 5-8 of HIC,
odd fractions from 1735th of gel filtration were used to perform SDS-PAGE. 2 SDS PAGE
(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) gels were prepared.