1. Introduction and objectives

1.1 Introduction 2

1.2 Objectives 7

1.3 Methodologies 8

1.3.1 Kinetics of inclusion body formation during protein expression 8

1.3.2 Analysis of structural and morphological characteristics of 8

inclusion body aggregates

1.3.3 Comparative solubilization of inclusion bodies in different 9

solubilizing agents and their effect on protein conformations.

1.3.4 Purification and characterization of r-enolase, r- Cu-Zn SOD 9

and L-asparaginase II.

1.3.5 Denaturation kinetics and effect of polyols on thermal 9

aggregation of recombinant L-asparaginase II.

1.4 References

1

1. Introduction and objectives

1.1 Introduction

Proteins are major structural and functional molecules of the living system.

They are required to be present in highly purified form for structure and functional

studies as well as for industrial applications. Before the advent of recombinant

DNA technology, proteins were usually being· recovered from their natural

sources by employing various purification techniques. It was very cumbersome

and time consuming process, often resulting in very low yield. Presently this

problem has been overcome by use of recombinant DNA technology that led to

the cloning and expression of almost any protein coding gene. However,

heterologous expression of proteins in host cells gave rise to another problem

due to the formation of protein aggregates inside the host cells. As proteins must

be present in their native conformation for structural and functional studies,

recovery of active proteins from these aggregates is inevitable.

Accumulation of protein molecules, apparently amorphous aggregates as

inclusion bodies (IBs) often occurs during expression of heterologous or some

endogenous proteins in Escherichia coli (1, 2). Frequently, recombinant

polypeptides are not able to fold properly within the host cells and form

aggregates consisting of misfolded proteins. These aggregates are known as

inclusion bodies or refractile bodies since they appear highly refractile w~en cells

are observed microscopically (3, 4). Formation of IBs occurs mostly in bacterial

cytoplasm but in case of secretory proteins it also takes place in perip~~mic ' ........ :\.

space (5, 6). Inclusion body aggregates, even though composed of recombiri'$r:tt ', '

protein molecules, also contain other molecules like nucleic acids, chaperones,

lipids, membrane proteins and sugars in low amount (7 -1 0). Formation of IBs

occurs due to inefficiency of bacterial folding machinery, unavailability of

chaperones, higher concentration of newly synthesized polypeptides, absence of

post-translation machinery and reducing environment of cytoplasm of E. coli (7).

Generally, hydrophobic interaction is considered to be dominant force in causing

aggregation of partially folded protein molecules formed during expression (11).

However, the formation of IBs mainly depends on the kinetic competition between

protein-specific folding and aggregation rates with synthesis rate of the

2

expressed protein (12). Inclusion body formation is a predominant feature of very

strong promoter systems. It is favored with high inducer concentration, with the

use of complex growth media and at higher cultivation temperature (13, 14). It

has also been suggested that the IB formation depends on the specific folding

behavior rather than on the general characteristics of a protein such as size,

fusion partners, subunit structure and relative hydrophobicity (12). However,

folding-rate-limiting structural characteristics as disulfide bonds and certain point

mutations can significantly promote the formation of aggregates (15, 16).

Inclusion bodies are imbibed with unique physical and structural properties

that make them very amenable and useful during large scale purification of

recombinant proteins. Inclusion bodies are generally denser than other cellular

molecules with density between 1.3-1.4 g/ml (17). Morphologically they appear

spherical, ovoid or cylindrical in shape with smooth as well as rough surface

architecture (18, 19). Structurally, they are mainly composed of disordered

misfolded molecules but recent studies using ATR-FTIR revealed the presence of

native-like secondary structures in substantial amount (20). Surprisingly, the

complete amorphous nature of IBs, as earlier thought, has also been challenged

by recent observations that have shown the presence of amyloidogenic property

(Congo-Red and Th-T binding) in IBs attributed by large percentage of ordered 13- . structure (21-24). Inclusion body aggregates are quite resistant to proteolytic

degradation in comparison to native protein molecules. They show reversible

dissociation and association of protein molecules and are highly dynamic in

nature in cytoplasmic milieu of E. coli (25). Besides these properties, role of

inclusion body aggregates in triggering amyloid like toxicity has been recently

observed (26).

The general procedure used for recovery of bioactive proteins from

inclusion bodies involves four basic steps: isolation of inclusion bodies;

solubilization of IBs aggregates; refolding of solubilized proteins and their

purification. Among these steps, solubilization and refolding are crucial steps that

affect the recovery of bioactive protein from IBs. Low recovery of bioactive

proteins results due to complete loss of secondary structure of inclusion body

proteins during solubilization which favors aggregation during refolding step. As

inclusion bodies formation is highly specific, they are mostly composed of

3

expressed recombinant proteins. Their isolation from cell lysates using proper

detergent and salt based washing methods can result in recovery of much

enriched homogeneous 18 preparation (27). This may help in reducing the

number of steps required during purification of the refolded proteins. In general,

proteins expressed as inclusion bodies are solubilized using high concentration

(6-8 M) of chaotropic solvents. Chaotropic agents such as urea, guanidine

hydrochloride, and thiocyanate salts (12, 28, 29) detergents such as SDS (30) N­

cetyl trimethyl ammonium chloride (31) and sarkosyl (sodium N-lauroyl sarcosine)

(32) along with reducing agents like ~-mercaptoethanol, dithiothreitol or cysteine

have been extensively used for solubilizing the inclusion body proteins. Besides

these solubilizing agents, use of reducing agent like OTT; ~-mercaptoethanol

along with chaotropes improves the solubilization yield of IB proteins. They help

in maintaining cysteine residues in reduced state and thus prevent non-native

intra or inter disulfide bond formation in highly concentrated protein solution at

alkaline pH. Chelating agents like EDTA are frequently used in the solubilization

buffer to prevent metal catalyzed air oxidation of cysteines.

Use of extreme pH with combination of low concentration of denaturing

agent or temperature has also been used for solubilization of inclusion body

proteins (27, 29, 33). A recent report also establishes the use of high hydrostatic

pressure (1-2 kbar) along with reducing agent for solubilization of IBs (34). Thus

studies of the behavior of these solubilization agents will certainly improve our

understanding of the forces involved in protein aggregation during inclusion body

formation (35). Such information can be used judiciously in development of an

improved method for inclusion body solubilization. A sparse matrix based

solubilization approach has been described for solubilization of inclusion body

proteins with the understanding of interaction involved in protein aggregation

using different buffer compositions (36). In case of very high level of protein

expression, in situ solubilization of inclusion body by directly adding denaturant

into the fermentation broth has also been reported (37). The main advantage of

this method is the elimination of mechanical disruption method with centrifugation

step for the recovery of inclusion bodies and thus may help in increasing the

overall yield of the recombinant protein. Thus the choice of the solubilizing agent

greatly affects the refolding yield and cost of the overall process.

4

Solubilized proteins are refolded into their native state by removal of the

chaotropic agents and other salts by dialyzing them in buffers containing reducing

and oxidizing agents (12, 29, 38). Purification of the recombinant protein either

can be carried out before renaturation or after refolding of the solubilized protein.

Refolding followed by purification is generally preferable as some of the high

molecular aggregates along with the contaminants can be removed in single step.

In spite of several protocols available for protein solubilization and refolding, the

overall recovery of bioactive protein from inclusion body is often very low. Dilution

of solubilized protein directly into the renaturation buffer is the most commonly

used method for small scale refolding of recombinant proteins. This helps in

reducing protein aggregation. In general, protein concentration is kept around 20-

50 IJg/ml to achieve better refolding yield. Refolding large amount of recombinant

protein using dilution method needs large refolding vessel, huge amount of buffer

and additional concentration steps after protein renaturation and thus adds to

high cost of protein production (39-41 ). Pulse renaturation involving addition of

small amount of solubilized protein to the renaturation buffer at successive time

intervals helps in reducing the volume of buffer thereby improves the overall

performance of the refolding process (41). Success of this process is based on

the fact that once a small amount of denatured protein is refolded into native form,

it does not form aggregates with the denatured proteins. Thus, by choosing the

appropriate protein concentration and time of successive addition of solubilized

protein, large quantities can be refolded in the same buffer tank. Pulse

renaturation processes have been successfully tried for the recovery of gamma

interferon and lysozyme (42, 43). Proteins consisting of large number of cysteine

residues often form random disulfide bonds amongst incorrect pairs of cysteines.

This, finally, leads to the aggregation of these proteins during refolding step of

purification. Generally, such proteins are refolded in presence of thiol groups

exchanging molecules like glutathione; cysteine, DTT/GSSG (44-46) and

oxidizing metal ions like Cu++ (41 ).

One of the major problems associated with the recovery of the refolded

protein from the solubilization mixture is its aggregation (47, 48). Aggregation is a

higher order reaction where as refolding is a first order reaction. Thus at high

initial protein concentration, the rate of aggregation is more than that of refolding

(49). Because of this kinetic competition, the yield of correctly folded protein

decreases at increasing protein concentration. Protein concentration in the range

of 10-50 IJg/ml is typically used during refolding (29, 41 ). Reduction in the extent

of protein aggregation and thus improvement in the yield of the refolded protein

has been achieved by use of monoclonal antibodies, chaperones and foldases

during refolding (50-52). Renaturation of bioactive protein with reduced

aggregation has also been achieved by addition of low molecular weight additives

(53). Often, additives such as acetone, acetamide, urea, detergents, sugars,

short chain alcohols, dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG)

are used to enhance the yield of folded bioactive protein (54). The most

commonly used low molecular weight additives have been L-arginine, low

concentration of urea or guanidine hydrochloride (1-2 M) and detergents (41).

The exact mechanisms of action of the low molecular weight additives are not

precisely known but they have been found suitable for refolding of many proteins

(55). These additives may influence both the solubility and stability of the

unfolded protein and folding intermediates (56). These are easily removed from

solubilization buffer except detergents, which need special treatment after protein

refolding. In most of the refolding methods, addition of renaturing agent or optimal

buffer or conditions (usually low temperature) helps in reducing the aggregation

of the protein intermediate. As propensity of aggregation reduces with low

concentration of protein, most of the time refolding is carried out in dilute

condition.

After purification, it is very important to keep protein for long period in its

active form for further use. There are various parameters used to improve the

stability of proteins. Still, it is not clearly understood why some substances when

added in protein solution do stabilize the protein structure. However, it has been

observed that these stabilizers somehow affect the physical properties of

solvents which finally help in stabilization of native structure of proteins. Co­

solvents like sugars, amino acids and salting out agents act through increasing

surface tension of solvent while polyols such as PEG act through preferential

exclusion mechanism (57-61).

Purification of proteins from Inclusion bodies involves four basic processes;

inclusion body isolation from cells, solubilization, refolding and protein purification

6

by various chromatography methods. Thus, an enhancement in yield of bioactive

proteins from inclusion bodies requires careful analyses and optimization of all

the four steps. By integrating the information acquired during each of the above

four steps, an optimal condition can be designed for high throughput recovery of

bioactive proteins from inclusion bodies.

1.2 Objectives

The main objective of this study was to understand the recombinant

protein aggregation during expression in E. coli and to develop methods for high

throughput protein purification from IBs. Four different IB forming proteins viz. E.

coli L-Asparaginase II, human growth hormone (hGH), human Cu-Zn Superoxide

dismutase (SOD) and Mycobacterium tuberculosis r- Enolase were used as

model proteins for the above studies. Detailed studies on mechanism of 18

aggregate formation during expression were carried out. Differences in

biophysical properties of in vivo to that of in vitro aggregates were studied.

Inclusion bodies were purified and .solubilized with different solubilizing agents.

EffeCt of novel mild solubilization agents (p-mercaptoethanol and n-propanol) on

protein secondary structural elements were studied in detail. Kinetics of unfolding

and refolding of purified recombinant proteins was studied to get a better

understanding of the protein folding mechanisms.

To achieve the objectives, the following research works were carried out

pertaining to inclusion body proteins produced in E.coli:

1. Kinetic of inclusion body formation during protein expression.

2. Structure and functional analyses of inclusion body proteins

3. Comparative solubilization of inclusion body proteins in different solubilizing

agents and their effects on protein conformations.

4. Purification and characterizations of r-enolase, r- Cu-Zn SOD and L­

asparaginase II from inclusion bodies.

5. Studies on denaturation kinetics ofrecombinant L-asparaginase II.

7

1.3 Methodologies

To achieve the above objectives, recombinant proteins were expressed as

inclusion bodies using E. coli as a host. Details of the different experiments used

to achieve the objectives are given below:

1.3.1 Kinetics of inclusion body formation during protein expression

1) Expression of Asparaginase, Human growth hormone, Superoxide dismutase

and Enolase as inclusion bodies in E. coli.

2) Analysis of protein expression at different stages of growth and isolation of

inclusion bodies after different time of induction.

3) Purification of inclusion bodies using sucrose density gradient ultra

centrifugation method and detergent washing method.

4) Size and shape analyses of inclusion body aggregates by particle size

analyzer and electron microscopy.

1.3.2 Analysis of structural and morphological characteristics of inclusion

body aggregates

1) Architectural studies of inclusion body proteins by proteolytic degradation.

2) Solubilization profile of inclusion body proteins in different concentration of

urea.

3) Amyloid behavior of inclusion body aggregates using Congo-Red and Th-T

dye binding assays.

4) ATR-FTIR study of purified IBs for determination of secondary structural

elements of protein present in IBs.

5) Electrical properties and charge distribution on inclusion body aggregates

using Zeta potential measurement.

8

1.3.3 Comparative solubilization of inclusion bodies in different

solubilizing agents and their effect on protein conformations.

1) Solubilization of inclusion body aggregates in urea, GdmCI, high pH and

organic solvents.

2) Effect of denaturants on structural elements of proteins by spectroscopic

methods.

3) In-vitro mixed aggregate formation and analysis of specificity of aggregation

by density gradient ultracentrifugation.

1.3.4 Purification and characterization of r-enolase, i- Cu-Zn SOD and L­

asparaginase II.

1) Solubilization and refolding of enolase, Cu-Zn SOD and L-asparaginase II.

2) Purification of these proteins using various chromatographic techniques.

3) Characterization of purified proteins by activity assays and spectroscopic

methods.

1.3.5 Denaturation kinetics and effect of polyols on thermal aggregation

of recombinant L-asparaginase II.

1) Denaturation kinetics of L-asparaginase in different buffers using CD and

fluorescence spectroscopy.

2) Study of effects of polyols on thermal aggregation of L-asparaginase. t~)

9

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