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

100

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.

101

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.

102

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

103

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.

104

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

105

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.

106

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

107

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,

108

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.

109

(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.

110

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.

111

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.

112

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)

120

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.

114

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

115

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

116

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.

117

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.

118

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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