4. Characterization of protein aggregates in...
Transcript of 4. Characterization of protein aggregates in...
4. Characterization of protein aggregates in
inclusion bodies
4.1 Introduction 101
4.2 Materials and methods 103
4.2.1 Chemicals and reagents 103
4.2.2 Cloning, expression and isolation of inclusion bodies 103
4.2.3 Solubilization of purified inclusion bodies 103
4.2.4 Size analysis of inclusion body aggregates 103
4.2.5 Proteolytic digestion of inclusion bodies 104
4.2.6 Amyloid specific dye binding assays of inclusion bodies 104
4.2.7 ATR-FTIR spectroscopy of pure inclusion bodies 105
4.3 Results and discussion 105
4.3.1 Kinetics of inclusion body formation 107
4.3.2 Solubilization and proteolytic susceptibilities of 18 proteins 111
4.3.3 Specificity of inclusion bodies to amyloid specific dyes 116
4.3.4 Structural analysis of inclusion bodies by FTIR spectroscopy 119
4.4 Conclusions 124
4.5 References 126
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4. Characterization of protein aggregates in inclusion bodies
4.1 Introduction
High level expression of heterologous proteins in E. coli often leads to
deposition of proteins in the form of insoluble aggregates defined as inclusion
bodies (IBs) (1-2). They are often present in cytoplasm or periplasm of
expression hosts and seen as dense refractile aggregates under electron
microscope (3-5). Aggregation of proteins during high level expression is,
generally, thought to be caused by high local concentration of na·lve polypeptide
chains emerging from ribosomes, inefficient folding and un-availability of
chaperones (6, 7). These factors lead to the formation of partially folded or
misfolded intermediates in cell cytoplasm. Usually, these intermediates consist of
surface exposed hydrophobic patches which can associate together to form large
amorphous aggregates as IBs (8). Although IBs have been characterized as
amorphous aggregates, they are not just cluster of misfolded intermediates (9-11 ).
Rather, they are often enriched in 13-sheet structures ( 12-15) and binds to amyloid
specific dyes like Thioflavin-T and Congo-Red (14, 16-18). They show seeding
behavior like amyloid fibrils assembly (14). Since, inclusion bodies mostly consist
of expressed proteins; their aggregation can be considered to be specific like
amyloids (19). Though inclusion body formation is considered to be irreversible
process, the study published by Carrio et a/. points out the reversible nature of
inclusion body formation where aggregation and solubilization of proteins occurs
simultaneously during expression (20). Existence of native-like structure of
proteins in inclusion bodies has also been observed (12, 13, 21). Besides this,
there have been reports suggesting the presence of biological activities of
proteins in inclusion bodies (22-24 ).
There are two proposed models which describe that the inclusion body
formation is a consequence of the self assembly of non-native monomers into
growing polymers of higher sizes. In first model, aggregation proceeds from a
single or limited number of nucleation sites by accumulation of the misfolded
intermediates. Since these nucleation aggregates are thermodynamically stable
entities, the addition of misfolded monomers on these aggregates are favored.
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Thus, it reduces the chance of the formation of new nucleation sites and leads to
the formation of giant IB aggregates. The second model treats IBs as aggregate
of aggregates in which small size aggregates tend to associate themselves to
give rise to one or more bigger aggregates (25) .
(a) ~ (b)
(9 ~ ~ \ ---,
~ .4 ~
{ / \ I • \ I
~ ~ #) -trendS" Gel/ Bootogy
Figure 4.1 Two models of inclusion body formation. (a) Protein aggregation (blue arrows) directly deposits aggregated protein into an inclusion body. Monomers are assumed to diffuse from their site of synthesis (or denaturation) to their site of deposition. (b) Aggregation of monomers occurs in the cell periphery, possibly by growth on an oligomeric seed. Small aggregates are then delivered (black arrows) by either diffusion or directed transport to the nascent inclusion body.
In this chapter, kinetics of inclusion body formation inside the E. coli cells
was studied and characteristics of protein aggregates were analyzed. E. coli L
asparaginase II and human growth hormone (hGH) were used as model proteins.
Physical and structural characteristics of inclusion body aggregates, like size
distribution, solubilization profile, amyloidogenic characteristics and protein
secondary structure were studied in detail. The objective was to understand the
structural aspects of inclusion body proteins so that an improved refolding
process can be developed.
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4.2 Materials and methods
4.2.1 Chemicals and reagents
Materials used in this study were similar as mentioned in chapter three
except urea, proteinase K, Thioflavin-T and Congo Red dyes were from Sigma
Chemical Ltd., USA.
4.2.2 Cloning, expression and isolation of inclusion bodies
Cloning, expression, isolation and purification of asparaginase and hGH
IBs were carried out using procedures mentioned in chapter three. Kinetic
analysis was carried out using IBs isolated and purified from the E. coli cultures
harvested at different time points (1, 2, 3 and 4 hours) after induction. Inclusion
bodies were purified to homogeneity using sucrose density gradient method as
described in chapter three. Purified inclusion bodies were used for the study.
4.2.3 Solubilization of purified inclusion bodies
Solubilization profile of pure inclusion bodies in urea was determined by
measuring turbidity of solubilized samples at 350 nm. Isolated hGH and
asparaginase IBs were solubilized in 50 mM Tris-HCI, 5 mM DTI, pH 8.5 buffers
containing different molar urea (0-8 molar) and left overnight. After solubilization,
· turbidities of samples were measured at 350 nm by spectrophotometer (UV 2450,
Shimadzu, Japan).
4.2.4 Size analysis of inclusion body aggregates
Asparaginase and hGH inclusion bodies, isolated at different time points
after induction of E. coli cells, were homogenized and their size distribution was
analyzed by Malvern mastersizer hydro 2000S (Malvern Instruments, USA): This
method is based on laser diffraction techniques in which the angles of scattering
for different sized particles are recorded. Then software converts this raw data
into % volume Vs size distribution graph for analyses purpose which can also be
converted into% number Vs size plot. Inclusion body samples were injected into
detection chamber with 1 0 % obscurity and were scanned 10 times. Size
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distributions of inclusion bodies were analyzed by plotting a graph between
percentage population and their size.
4.2.5 Proteolytic digestion of inclusion bodies
Purified hGH and asparaginase inclusion bodies, obtained after different
time points of induction, were subjected to proteolytic digestion. Inclusion bodies
were diluted to 1 00 at 350 nm in 980 IJI of 50 mM Tris-HCI, 150 mM NaCI buffer
of pH 8.0. Proteolytic digestion of IBs was initiated by adding 20 IJI proteinase K
stock (0.2 mg/ml) to the inclusion body solution (at 4 IJg/ml final concentration).
Proteolytic digestion was monitored for 200 minutes (hGH IBs) and 100 minutes
(Asparaginase IBs) by measuring the changes in OD at 350 nm in UV-2450
spectrophotometer (Shimadzu, Japan). Enolase and SOD inclusion bodies were
purified as described in chapter 3 and used for proteolytic digestion.
4.2.6 Amyloid specific dye binding assays of inclusion bodies
Asparaginase and hGH IBs were tested for Congo red (CR) binding by the
spectroscopic band shift assay (14). Inclusion bodies were diluted in reaction
buffer (10 mM sodium phosphate of pH 7.0, 150 mM NaCI) containing 10 IJM CR
to final protein concentration of 25 IJg/ml. Samples were incubated for 10 minutes
at room temperature before acquisition of spectra. Absorption spectra were
collected together with negative control solutions of dye in the absence of protein
and of protein in absence of dye on a UV-2450 spectrophotometer (Shimadzu,
Japan). Individual scattering contribution of IBs spectra was subtracted from their
respective samples spectra (dye+ protein).
The final reaction mixture for thioflavin-T (Th-T) assay consisted of 50 IJg/
ml protein, 10 mM phosphate buffer of pH 7.0, 150 mM NaCI, and 75 IJM Th-T.
Samples were kept at room temperature for 10 minutes for thermal equilibration.
Fluorescence emission spectra were recorded from 460 to 600 nm using an
excitation wavelength of 440 nm (Cary Eclipse spectrophotometer, Varian,
Australia). 5 nm slit width was fixed for both excitation and emission wavelength.
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4.2.7 ATR-FTIR spectroscopy of pure inclusion bodies
Asparaginase and hGH inclusion bodies were purified by sucrose density
gradient method. These IBs were lyophilized to remove water. Samples were
dried in a Speed-Vac system for 2 hours. The dried samples were spread on
BaF2 crystal cells for spectra acquisition. The structure of dry IBs aggregates was
analyzed directly in Bruker Tensor FTIR spectrometer. For each spectrum (1000
cm-1 to 4000 em-\ 25 interferograms were collected and averaged. Second
derivatives of the amide I & II region spectra were used to determine the
frequencies at which the different spectral components were located. These
frequencies were used for assignment of secondary structural contents in
inclusion body proteins.
4.3 Results and discussion
The formation of insoluble aggregates of recombinant proteins during high
level expression is driven by the association of partially folded or misfolded
intermediates. These aggregates have been thought to be resistant to proteolytic
cleavage. However, there are studies which indicate their susceptibility to
proteases. It is thought that IBs are amorphous aggregates without any orderly
arrangement of aggregating species. Importantly, still there are no conclusive
studies which deal with roles of sequential patterns, variations in sizes and amino
acid sequences in inclusion body formation. However, on the basis of physical
characteristics two types of inclusion body aggregates had been observed. One,
which is compact in nature and resistant to denaturants while other is soft and
relatively more labile. The tough aggregates are called classical IBs where as
soft IBs are defined as non-classical IBs. Not much structural information is
available on non-classical IBs except their solubility in low concentration of
denaturants (26, 46).
To address this question, proteolytic susceptibility of asparaginase,
enolase, hGH and SOD inclusion bodies were analyzed. The results obtained
from this study indicated differential susceptibilities of these IBs to proteinase K
(Figure 4.2). Inclusion bodies of hGH were found resistant to proteinase K
whereas asparaginase IBs were highly prone to degradation. In 20 minutes 80 %
of asparaginase IBs was degraded whereas only 20 % degradation was
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observed for hGH IBs. Degradation profile of enolase and SOD inclusion bodies
were in between hGH and asparaginase. This apparently indicated the non
classical nature of asparaginase inclusion bodies. This was confirmed from the
denaturation profile of hGH and asparaginase IBs in different molar urea
solutions (Figure 4.3). Complete solubilization of hGH IBs occurred at 7 molar
urea concentration (Figure 4.3a). However, 2 molar urea was found to be
sufficient for complete solubilization of asparaginase IBs (Figure 4.3b). Thus,
these studies revealed hard and compact nature of hGH IBs and soft and loose
nature of asparaginase IBs. It can be concluded that hGH inclusion bodies are of
classical nature whereas asparaginase IBs are of non-classical type.
These observations raised the following questions: how these differences
originate during expression of different proteins? What are the physical,
biochemical and structural basis of differences between these aggregates? To
address these questions, size distribution, TEM, solubilization profile of
aggregates, proteolytic degradation, Congo-Red and Th-T binding assays of IBs,
isolated from culture harvested at different time points after induction were
carried out. FTIR Spectra of the pure IBs were also acquired to understand the
secondary structural contents of inclusion body proteins.
hGH E 1.0 ___.,__ Enolase c::
0 ----<J-- SOD LO (V)
0.8 ----Asparaginase II -C'O Q) () c:: C'O 0.6 .0 .... 0 C/) .0 <(
0.4 "0 Q)
.!:::! C'O E 0.2 .... 0 z
0 10 20 30 40 50 60 70 Time (minutes)
Figure 4.2 Kinetics of proteolytic digestion of inclusion body aggregates by Proteinase K.
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1 2 3 4 5 6 7 8 9 M 12 3 4 56 7 8 9M
--(a)
Figure 4.3 SDS-PAGE of solubilized inclusion body supernatants. (a) hGH ISs. Lane 1-9, supernatants of 0 to 8 molar urea. (b) Asparaginase ISs. Lane 1-9, supernatants of 0 to 8 molar urea; lane M, LMW marker (97, 66, 45, 30, 20.1 and 14.4 kDa).
4.3.1 Kinetics of inclusion body formation
The size distribution of protein aggregates during inclusion body formation
in E. coli is not clearly understood yet. Although , proteins in inclusion body
aggregates have native-like structure, the exact mechanism of nucleation and
growth phase of inclusion body formation is not well understood. To address this
question, sizes of ISs isolated from E. coli cells harvested at different time points
(1 , 2, 3 and 4 hours duration) after induction were analyzed . Ultra pure ISs from
these cultures were isolated by procedures as described in the methods section.
Size distribution patterns of hGH and asparaginase ISs analyzed by particle sizer
are presented in figure 4.4. It was observed that asparaginase ISs sizes were
independent of post induction periods whereas sizes of hGH ISs increased
progressively and saturated around 4 hours after induction. The size distribution
pattern of asparaginase ISs was remained constant for four hours duration after
induction (Figure 4.4a). There were no distinct nucleation and growth phases
present within shortest possible time limit of 60 minutes after induction. This was
also supported by transmission electron micrographs of these ISs (Figure 4.5a).
Size of pure asparaginase ISs, isolated after different time of IPTG induction,
were almost same [Figure 4.5a (1 , 2, 3 and 4)]. However, there were
approximately 15 % aggregates in range of 20-40 nm in inclusion body
preparations of different post induction periods. It could be interpreted that these
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14
12 • ASN 1 hr
10 • ASN 2hrs ... ASN 3hrs ..-:::R • ASN 4hrs 0 8 -....... Q)
..0 E 6 ::J z
4
2
0
0.0 0.2 0.4 0.6 0.8 1.0 (a) Size (micrometer)
14
12 hGH 1 hr hGH 2 hrs
10 hGH 3 hrs ..-
hGH 4 hrs :::R 8 0
....... Q)
..0 6 E ::J z 4
2
0
-2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
(b) Size (micrometer)
Figure 4.4 Size distribution patterns of IBs isolated from E. coli cells harvested at different time points after induction. (a) Asparaginase IBs. (b) hGH IBs. Colored bars indicate the harvesting time.
smaller aggregates either form in newly formed daughter cells or it may arise due
to continuous seeding mechanism happening in cells during expression. The
expression level of asparaginase saturated only after four hours of induction
while saturation in sizes of IBs happened within 60 minutes after induction. Thus,
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the size of aggregates could have increased after 60 minutes of induction if newly
synthesized polypeptide chains would have been deposited on the same seed in
a single cell. But it was contrary to size distribution result obtained for
asparaginase IBs. So, it can be concluded that multiple nucleation seed were
formed in a single cell of E. coli during expression of recombinant asparaginase.
Growth of these seeds occurred for short duration and size got saturated within
30 minutes and further new seeds were formed during expression period. Most of
the asparaginase IBs were in range of 100-200 nm sizes. Formation of similar
size aggregates for asparaginase IBs at different time points after induction was
further confirmed from TEM studies (Figure 4.5a). The transmission electron
micrograph of E. coli cells expressing asparaginase showed single dense area at
proximal ends. It could be possible only if these small size IBs associate loosely
and form bigger size aggregate. However, they must have been dissociated
during sonication as size analysis indicated.
In contrast, the size distribution pattern of hGH inclusion bodies formed
after induction for different durations, indicated the presence of distinct nucleation
and growth phases during 18 formation (Figure 4.4b). The maximum size of IBs
after 4 hours of the induction point was in range of 0.3 to 1 IJm. Seeding started
immediately after induction and there was gradual increase in size of 18
aggregates till 3 hours of post induction period (Figure 4.4b). The increase in size
of 18 aggregates was highest between 2-3 hrs of post induction period. There
was no difference in size distribution patterns of IBs isolated at 3 and 4 hours
time points after induction. It may occur due to the decrease in rate of protein
synthesis as cells enter stationary phase of growth. After one hour of induction,
90% of 18 aggregates had smaller size (25-150 nm) (Figure 4.4b). With increase
in post induction period the population of these smaller size IBs was reduced to
65 % at 2 hours and at the end of 3 hours after induction merely 10 % 18
aggregates were smaller than 150 nm. It indicated that hGH inclusion body
formation starts immediately after induction, where seeds are formed and they
grow in size continuously during expression period. Increase in size of hGH
inclusion bodies at different time points after induction was also confirmed by
TEM analyses (Figure 4.5b). This phenomenon was entirely different from what
was observed for asparaginase inclusion bodies.
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(b)
Figure 4.5 Transmission electron micrograph of purified inclusion bodies. (a) Asparaginase and (b) hGH. 1, 2, 3 and 4 are IBs isolated after 1, 2, 3 and 4 hours of IPTG induction. Bar represents 2 1-Jm.
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Thus from above study, it can be concluded that different proteins follows
different aggregation mechanism during recombinant protein expression in E. coli.
Expression of asparaginase in E. coli leads to the formation of large number of
aggresomes (small aggregates) in a single cell which loosely associate amongst
themselves to form bigger size IBs. While expression of hGH IBs in E. coli results
in the formation of single large aggregate in a cell by continuous deposition of
protein molecules on a nucleation seed. The proteolytic digestion and
solubilization profile of hGH and asparaginase IBs were in agreement with size
distribution pattern. Tough and large IBs are formed by sequential deposition
whereas soft and smaiiiB aggregates are formed immediately after induction.
4.3.2 Solubilization and proteolytic susceptibilities of IB proteins
To measure the extent of compactness of protein aggregates in inclusion
bodies, purified IBs were subjected to different denaturing conditions and
proteinase K digestion. Solubilization profile of hGH and asparaginase inclusion
bodies in different molar concentrations of urea was determined by measuring
. the turbidity of supernatants at 350 nm (Figure 4.6) . It was observed that hGH
inclusion bodies were progressively more soluble with increasing urea
concentration from 1-8 M. Complete solubilization of inclusion body aggregates
occurred at 7 M urea solution. Most interestingly, the solubility of proteins from
inclusion bodies isolated at early hour after induction was more in comparison to
those isolated at 2-4 hours after induction (Figure 4.6a). For asparaginase, the
solubilization pattern of protein in different molar concentrations was independent
of harvest time after induction (Figure 4.6b).
This difference in solubilization profile of hGH inclusion bodies, isolated from
cells harvested at different time points after induction, may be due to differences
in composition of IB aggregates. It was also observed that hGH IBs, isolated after
1 hour of induction, mostly consisted of smaller size aggregates in comparison to
IBs isolated at 3 to 4 hours after induction. These smaller aggregates may get
solubilized at slightly lower concentration of urea owing to their large accessible
surface area. However, this possibility can be negated as sufficient time was
given for solubilization of these IBs. Thus, it can be inferred that increase in post
induction period makes hGH inclusion bodies more resistant to denaturants.
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1.2
• hGH 1 hr 1.0 • hGH 2 hrs
E ... hGH 3 hrs c 0.8 0 ... hGH 4 hrs I!) (")
..... 0.6 m
>-::: :"2 .Ll 0.4 ,_ :::J I-
0.2
0.0
0 2 4 6 8
Urea [M] (a)
1.2
1.0 • ASN 1 hr
• ASN 2 hrs E
0.8 ... ASN 3 hrs c 0 ... ASN 4 hrs I!) (") ..... 0.6 m >-:::
"0 0.4 :.0 ,_ :::J I-
0.2
0.0
0 2 4 6 8 Urea [M]
(b)
Figure 4.6 Solubilization profiles of inclusion bodies isolated from cell harvested at different time points after induction. (a) Asparaginase IBs. (b) hGH IBs. Colored bars indicate the harvesting time.
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Similar results were obtained from proteinase K degradation study (Figure
4. 7). Asparaginase inclusion bodies were very prone to proteinase K attack and
there was no effect of post induction time on susceptibility of inclusion bodies to
proteinase K (Figure 4.7a). Asparaginase inclusion bodies showed steep slope
for denaturant based solubilization and proteinase K digestion. This revealed that
the stabilizing forces between aggregating molecular species in asparaginase
inclusion bodies are weak and very unstable. The removal of aggregated
molecules from the asparaginase IBs surface during solubilization was rapid and
happened within very narrow range of urea concentration. The proteinase K
digestion required less time to degrade asparaginase inclusion bodies. However,
hGH inclusion bodies isolated after 1 hour of induction were more prone to
proteinase K attack and degradation in comparison to inclusion bodies isolated
after 2, 3, and 4 hours of post induction (Figure 4.7b). There was gradual
decrease in susceptibility of hGH IBs to proteinase K with increase in post
induction time. Human growth hormone inclusion bodies were more compact and
required higher concentration of denaturant for complete solubilization. Their
solubilization was slow and occurred in broad concentration range of urea. The
concentration of urea for 50 % solubilization of hGH inclusion bodies isolated at 1,
2, 3, and 4 hours of post induction were found to be 4. 7 M, 4.95 M, 5.2 M and 5.5
M respectively. This showed that the compactness and strength of interaction
between aggregating intermediates increased with increase in post induction time.
No such changes were observed in case of asparaginase inclusion bodies. Thus,
solubilization and proteolytic data impart that not only the size but also the quality
of asparaginase inclusion body aggregates do not change with increase in post
induction time.
Furthermore, the homogeneity in arrangement of protein molecules in
inclusion bodies was deduced by calculating the change in rate of proteolytic
degradation of IBs with time. It was of interest to know whether there is a
difference in the arrangement of protein molecules between core and peripheral
regions of IBs. The first derivative graph of proteinase K digestion of IBs revealed
that the rate of degradation decreased with reaction time (Figure 4.8a). This
could be either due to the compact nature of inner layers of IBs which got
exposed after removal of outer layers or it may result due to decrease in IB
11 3
concentration (as it act as substrate for proteinase K, continuous conversion of
inclusion body aggregates into degraded fragments during reaction will decrease
the substrate concentration, thus reaction rate). To find out the possible reason
for this degradation pattern, rate of change in degradation rate was plotted with
respect to turbidity of inclusion bodies (350 nm) at different time points of reaction
(i.e. substrate concentration) (Figure 4.8b).
- 1.0 "0 Q)
.!:::! ro 0.8 E .... 0 c .._...
0.6 E c
0 I!)
0.4 (") -ro >--i5 0.2 :0 .... ::I f- 0.0
1.0 -"0 Q)
.!:::! 0.8 ro E .... 0 0.6 z E c 0.4 0 I!) (")
15 0.2
0 (a)
25 (b)
20
50
--oo-- ASN 1 hr --<I-- ASN 2hrs ___.........__ ASN 3hrs ____,,_ ASN 4hrs
40 60 80 100 Time (minutes)
--hGH 1 hr ~hGH 2 hrs ~hGH 3 hrs -- hGH 4 hrs
75 100 125 150 175 200 Time (Minutes)
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Figure 4.7 Proteinase K degradation profiles of inclusion bodies isolated from E. coli cells harvested at different time points after induction. (a) Asparaginase IBs. (b) hGH IBs. Colored bars indicate the harvesting time.
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0.01
0.00 E _1:.
c -0.01 r ,
0 1.0
---hGH IBs ("') - -0.02 ! ------------ ASN IBs ~
I--o -0.03 I -->. I :-!:: -o -0.04- • :.0 ' .._ • ::I
' - -0.05 <J • i
-0.06- v -0.07
' ' I
0 50 100 150 200 (a) Time (minutes)
>. 5 :-!:: :"Q
4 ..0 ----o- ASN I Bs .._ ::I I- 3 ~hGHIBs -o --- 2 E c
0 1 1.0 ("') - 0 ~
I- -1 -o -->. -2 :-!:: -o :.0 -3 .._ ::I -3 -4 <J
-5 0.0 0.2 0.4 0.6 0.8 1.0
(b) IBs Turbidity (350 nm)
Figure 4.8 Proteinase K degradation profiles of hGH and asparaginase inclusion bodies isolated after 4 hours of induction. (a) Rate of degradation of IBs with time. (b) Rate of change in degradation rate of IBs with decrease in turbidities.
The rate of change of degradation rate was negligible with respect to substrate
concentration. It can only be possible in an enzyme catalyzed reaction if
substrates (IBs) are similar in nature.
Thus, it can be concluded that the smaller aggregates which form during
proteinase K degradation have similar morphology and topology as intact IBs and
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there is no difference in arrangement of protein molecules between core and
peripheral regions of 18 aggregates. However, differences in arrangements of
protein molecules were observed among hGH IBs isolated at different time points
after induction.
4.3.3 Specificity of inclusion bodies to amyloid specific dyes
It has been observed that amyloid fibril interact specifically with Congo red
(CR) dye which depends on secondary structure of interacting entities of fibril
especially ~-pleated sheet conformation (27). In fact this ~-sheet conformation of
fibril appears to be the crucial factor in CR binding. Other proteins that lack or
contain only minor proportion of ~-sheet structure do not stain with CR (28-30).
The interaction of fibril with CR also induces shift in the characteristic spectrum of
CR which depends on the nature of aggregate conformation. Inclusion body
aggregates also consist of less ordered secondary structures. There are several . reports which indicate the binding of the IBs with CR dye (14, 16-18). Therefore,
binding properties of IBs with CR was analyzed to check their amyloidogenic
properties. CR alone exhibits absorption maxima at 490 nm that shift to red , once
it binds to amyloid material. Asparaginase and hGH IBs isolated at different time
point after induction promoted a strong red shift in the absorption maxima (Figure
4.9a and 4.9b). The difference spectra showed broad peaks at 565 nm for
asparaginase IBs and at 560 nm for hGH IBs. However, absorbance of CR with
IBs isolated at early time point of post induction was less in comparison to that of
inclusion bodies harvested at later time points. The absorbance of CR bound
hGH IBs was higher than that of asparaginase IBs. It revealed that hGH IBs are
more amyloidogenic in nature in comparison to asparaginase IBs.
This result is also supported by thioflavin-T (Th-T) binding, which is an
amyloid specific fluorescent dye. It also undergoes characteristic spectral
alteration on binding to a variety of amyloid fibril, which does not occur on binding
to the precursor polypeptides, monomers, or amorphous aggregates of peptides
and proteins (31 , 32). Binding of amyloid fibril with Th-T alters excitation and
emission maxima of dye. In presence of fibril , it shows excitation maxima at 440
nm and emission maxima at 482 nm. When it binds to amyloid fibril there is large
enhancement in fluorescence intensity of Th-T relative to free dye. The binding of
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Th-T dye with hGH IBs brought sharp increase in fluorescence intensity in
comparison to asparaginase IBs at equal protein concentrations (Figure 4.1 Oa
and 4.1 Ob ). It was observed that increase in post induction period resulted in
0.5 CR alone
0.4 ASN 1 hr ASN 2 hrs
0.3 ASN 3 hrs Q) ASN 4 hrs u
0.2 c ro .0 ,_ 0 0.1 (/) .0 <(
0.0
-0.1
-0.2
400 450 500 550 600 650 700
(a) Wavelength (nm)
0.5 CR alone
0.4 hGH 1 hr hGH 2 hrs
0.3 hGH 3 hr Q) hGH 4 hrs u c
0.2 ro .0 ,_ 0 (/)
0.1 .0 <(
0.0
-0.1
-0.2 400 450 500 550 600 650 700
(b) Wavelength (nm)
Figure 4.9 Spectral characteristics of Congo-Red (CR) and difference spectra of IBs. (a) Asparaginase and (b) hGH inclusion bodies isolated at different time points after induction. Concentrations of CR and IBs used for assay were 10 1JM and 25 IJg/ml respectively.
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100
80 _........ :J -< .......... Q) 60 (.) c Q) (.) (/) Q) 40 ..... 0 :::J
LL
20
0
200
Q) (.)
~100 (.) (/)
~ 0 :::J
LL 50
ASN 1 hr ASN 2hrs ASN 3hrs ASN 4hrs
450 475 500 525 550 575 600 625 .
(a)
450
(b)
475
Wavelength (nm)
--hGH 1 hr -- hGH 2 hrs ____,.,___ hGH 3 hrs --- hGH 4 hrs
500 525 550 575 600 Wavelength (nm)
Figure 4.10 Spectral characteristics of Th-T binding with IBs. (a) Asparaginase and (b) hGH inclusion bodies isolated at different time points after induction. Concentrations of Th-T and IBs used for assay were 75 IJM and 50 IJg/ml respectively.
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formation of harder aggregates of hGH inclusion bodies which showed stronger
binding to Th-T dye. But, asparaginase IBs isolated at different time points after
induction had similar binding strength with Th-T. Emission maxima for hGH and
asparaginase IBs were around 475 nm and 485 nm. This small difference in
excitation maxima has been observed for different proteins.
However, the question arises whether this difference in CR and Th-T
binding to asparaginase and hGH inclusion bodies occurred due to variation in
arrangement of protein molecules inside inclusion body aggregates or it depends
on the proportion and types of secondary structural elements present in IBs. It
has been observed that CR binds to native proteins with various secondary
structures (33, 34) , as well as to partially unfolded proteins (33), protein
oligomers (35), and lipids (36). Th-T is generally considered to be more specific
towards amyloid fibrils than CR, and the characteristic fluorescence of ThT does
not generally occur upon binding to the precursor proteins or amorphous
aggregates of proteins (31, 32, 37, 38). There are, however, several exceptions
to this general rule, e.g., the fluorescent binding of Th-T to native AChE (39, 40).
In a few other cases, dimers, trimers, and larger aggregates of, for example, 13-
lactoglobulin cause fluorescence, indicating that Th-T binding is not restricted to
complete amyloid fibrils (41). Apart from proteins, Th-T has also been shown to
yield its characteristic fluorescence upon binding to DNA (42), y-cyclodextrin (40,
43, 44) and sodium dodecyl sulfate micelles (45). Thus, Structural analysis of
proteins in inclusion body aggregates was inevitable to explain the binding
characteristics of inclusion bodies with CR and Th-T dyes.
4.3.4 Structural analysis of inclusion bodies by FTIR spectroscopy
To find the possible role of secondary structural elements of hGH and
asparaginase IBs in differential binding properties to CR and Th-T dyes, their
FTIR spectra were analyzed. The native hGH is a predominantly helical protein
with small number of loops and turns, while asparaginase consists of both helices
and 13-sheets in equal ratios and large number of loops and turns. The analyses
of second derivative spectra of native and inclusion bodies of hGH showed that
formation of inclusion body aggregates lead to the increase in 13-sheet content
(4.11a and 4.11b). Second derivative spectra of hGH IBs showed prominent peak
119
area at 1654 cm-1 , characteristics of a-helices with small peak area at 1623 cm-1 ,
characteristic of 13-sheet. In spite of small proportions of 13-sheet content, hGH
inclusion bodies showed very strong binding with CR and Th-T dyes. It was also
found to be more resistant to proteolysis and denaturation. From the amino acid
sequence of helices forming regions of hGH protein, it was observed that majority
of them consist of hydrophobic amino acid residues (Figure 4.12).
0.02
-o.02 ::> -< -0.04
·0.06
-o.os
-o.1 +---~----.-----r-----.-----r--~ 1700 1680 1660 1640 1620 1600 1580
Wavenumber (cm-1)
0.0005
0.0000
.0.0005
.().0010
~-sheet
a-helix
1700 1680 1660 1640 1620 1600 Wave number (cm.1)
Figure 4.11 FTIR spectra of hGH. (a) Second derivative amide spectra of hGH in solution. 1, native hGH; 2, lyophilized in sucrose; 3, lyophilized in trehalose and 4, hGH dried alone (M.S. Salnikova et a/. International Journal of Pharmaceutics 358 (2008) 1 08-113). (b) Second derivative spectrum of hGH IBs.
(a)
>spjP01241jSOMA_HUMAN Somatotropin OS=Homo sapiens
MATGSRTSLLLAFGLLCLPWLQEGSAFP
TIPLSRLFDNAMLRAHRLHQLAFDTYQEF
EEAYIPKEQKYSFLQNPQTSLCFSESIPTP
SNREETQQSNLELLRISLLLIQSWLEPVQF
LRSVFANSLVYGASDSNVYDLLKDLEEGI
QTLMGRLEDGSPRTGQIFKQTYSKFDTN
SHNDDALLKNYGLLYCFRKDMDKVETFL
RIVQCRSVEGSCGF
(b)
Figure 4.12 (a) Crystal structure of native hGH (from PDB). (b) Amino acid sequence of hGH (Swiss-Prot Database). Red color highlighted regions indicate presence of hydrophobic residues involves in making hydrophobic and amphipathic helices.
120
These residues were present at regular intervals and formed amphipathic
helices. These helices may interact strongly and form very compact and hard
aggregates. So, it can be concluded from these observations that a-helices
present in aggregates may also bind strongly to these amyloid specific dyes. The
formation of compact and insoluble aggregates does not necessarily involve the
participation of beta sheets and may form by strong interactions between
amphipathic helices.
The FTIR spectra of Asparaginase IBs revealed the presence of large
proportion of beta sheet content in IBs in comparison to native proteins
(Figure4.13a and 4.13b). The crystal structure of Asparaginase also entails that
protein is very rich in beta pleated secondary structure (Figure 4.14b). Thus the
crystal structure of asparaginase and beta sheet content present in IBs, shown by
FTIR spectra (Figure 4.14a), were in accordance with its binding properties to
amyloid specific dyes.
{a}
• g ~ 0
! (b)
{c)
21100 . 11100 1800 1700. 1800 1500 1400
Wavenumbers (an·t
Broader peak in beta sheet region
2.01,-,---..--r--',.......,___,.---o+-o----.---.--,----,
Q) 1.6 (.) s::::
"' .Q 0 1.2 1/) .Q
~ 0.8
Asparaginase
1700 1680 1660 1640 1620 1600 wave m.mber (cm"1
)
Figure 4.13 (a) Amide band region for dried samples, as determined by FT-IR spectroscopy. Spectra shown are of (a) L-asparaginase freeze-dried alone, (b) freeze-dried in the presence of trehalose (1 .0 mg/ml) and (c) hydrated Lasp.araginase. All spectra have been normalized with respect to the amide I bands ( K.R. Ward eta/. : International Journal of Pharmaceutics 187 (1999) 156 153-162). (b) FTIR spectra of asparaginase inclusion bodies.
121
0.004 paraginase Q)
> n; o.oo2 > "i: ~ 0.000 "'C g -0.002 (.) Q)
(/) -0.004
1700 1680 1660 1640 1620 1600 Wave number (cm-1
)
(a)
(b)
Figure 4.14 (a) Second derivative spectrum of asparaginase ISs in amide band region . Broader peak in 1620-1640 cm-1 and 1680-1685 cm-1 range indicates large beta sheet content. (b) Crystal structure of native asparaginase (from PDB).
122
However, the denaturation and proteinase K degradation profiles indicate
that asparaginase ISs are very soft and prone to degradation. It could happen
due to irregular arrangements of beta sheet regions present in it, leaving large
accessible area to proteases and denaturants for action. Most importantly, the
enzyme activity, though low in comparison to native protein, was also observed in
asparaginase ISs.
All these studies entail the significant difference in the aggregation
behavior of recombinant asparaginase and hGH during inclusion body formation.
The physical and structural characteristics of these two ISs differed prominently
in their size, resistance to denaturants and proteases, and binding strength to
amyloid specific dyes. These variations may arise due to differences in their
nucleation and growth properties during ISs formation. However, the possible role
of amino acid sequences and intermittent topologies formed by association of
secondary structural elements present in folding intermediates (formed during
expression) in generation of these differences can not be ruled out.
Non-classical inclusion body (Asparaginase) proteins have following
characteristics:
1. Smaller in size(< 200 nm) and size do not vary with post induction time.
2. Less refractile in nature.
3. Soluble in lower concentration of denaturants.
4. Consist of native-like secondary structures.
5. Have residual biological activity.
6. Shows amyloid like properties.
7. Highly susceptible to protease attack.
123
4.4 Conclusions
These studies showed that there is fundamental difference in the
aggregation behavior, physical and structural characteristics of asparaginase and
hGH inclusion bodies. Kinetics of inclusion body formation and its size correlates
with solubilization profile of proteins from the aggregates. On the basis of the
solubility and presence of biological activity, inclusion bodies have been
categorized as classical (c) and non-classical (nc). The nciBs compared to
classical IBs are characterized by their higher fragility, higher solubility, and high
amount of correctly folded target protein or its precursor (46). Thus, our
observations indicated non-classical nature of asparaginase IBs and classical
nature hGH IBs. Following conclusions can be drawn from the kinetic and
structural studies of inclusion body proteins.
1. The mechanisms of inclusion body formation for recombinant
asparaginase and hGH were quite different. The seeding and growth
phases of asparaginase inclusion body formation were of shorter duration
and resulted in formation of small size (1 00-200 nm) aggregates in
comparison to hGH. However, hGH inclusion body formation started from
smaller nucleus and grown into bigger size with post induction period.·
2. Human growth hormone inclusion bodies were more resistant to
proteinase K and showed solubilization profile in urea like a classical
inclusion body.
3. Asparaginase IBs were more susceptible to denaturants and proteases
contrary to hGH. The post induction period had little effect of proteolytic
susceptibility and chaotropic resistivity of asparaginase IBs in comparison
to hGH. It showed that increase in post induction period made hGH IBs
more resistant to denaturants and proteases.
4. The molecular arrangements of misfoded or native-like polypeptides in
inclusion body aggregates are homogenous and uniform from inner core
region to peripheral region.
5. Inclusion body proteins have amyloid like properties as indicated by Congo
Red and Th-T binding assays.
124
6. Inclusion body proteins consist of higher percentage of beta sheet
conformation in comparison to native proteins.
7. The formation of hGH inclusion bodies during expression in E. coli
involves strong interactions between partially folded, amphipathic or
hydrophobic a-helices.
8. Both classical and non-classical inclusion body proteins have native-like
secondary structures.
9. Non-classical inclusion bodies are generally smaller in size and are less
refractile.
10. Proteins in non-classical inclusion bodies retain residual activity whereas
in classical inclusion bodies, they are completely devoid of any biological
activity.
125
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