Engineering of Proteinaceous Ligands
`
`for Improved Performancein Affinity
`
`Chromatography Applications
`
`Susanne Giilich
`
` ws
`\ KONST#SS)
`
`KTH
`
`RoyalInstitute of Technology
`Department of Biotechnology
`
`Stockholm 2002
`
`Page 1
`
`KASHIV EXHIBIT 1064
`IPR2019-00797
`
`Page 1
`
`KASHIV EXHIBIT 1064
`IPR2019-00797
`
`

`

`© Susanne Giilich
`
`Department of Biotechnology
`RoyalInstitute of Technology
`SE-106 91 Stockholm
`Sweden
`
`Printed at Universitetstryckeriet US AB
`Box 700 14
`100 44 Stockholm, Sweden
`
`ISBN 91-7283-248-7
`
`to
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`Page 2
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`Page 2
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`

`

`Susanne Giilich (2002): Engineering of Proteinaceous Ligands for Improved Performance in
`Affinity Chromatography Applications. Department of Biotechnology, Royal
`Institute of
`Technology, Stockholm, Sweden.
`
`ISBN 91-7283-248-7
`
`Abstract
`
`Affinity chromatography has proven to be a powerful unit operation allowing purification and
`concentration ofthe target protein in a single step thus, decreasing the number of consecutive unit
`operations. However, affinity chromatography may include some drawbacks. The objective ofthis
`thesis has been to use different protein engineering strategies for design of robust and predictable
`protein purification systems, addressing different problems faced in both large-scale and small-
`scale protein production.
`
`Oneofthe problemsin the recovery ofantibodies and their fragments by protein A-basedaffinity
`chromatography is the low pH, which is normally essential to elute the bound material from the
`column. Some antibodies are not able to withstand these conditions and suffer from irreversible
`inactivation. Here, this problem is addressed by constructing destabilized mutants of a domain
`analog (domain 7) from staphylococcal protein A. In order to destabilize the IgG-binding domain,
`two protein-engineered variants were constructed using site-directed mutagenesis of the second
`turn of this antiparallel three-helix bundle domain. In one mutant (Z6G), the second turn was
`extended with six glycines in order to evaluate the significance ofthe turn/loop length. In the other
`mutant (ZL4G), the original turn sequence was exchanged for glycines in order to evaluate the
`importance of the turn forming residues. By changing the properties of the turn the stability of
`both mutants was decreased, which could be used for breakage ofthe protein-ligand interaction at
`milder conditions. Hence, it is shown that turn/loop engineering may be anattractive approachto
`modulate a protein’s specific properties.
`
`One ofthe problems with proteinaceousaffinity ligands is their sensitivity to alkaline conditions.
`Manyapplications in the pharmaceutical and biotechnologicalfield, such as large-scale production
`of antibodies
`and albumin for
`therapeutic use,
`require extreme attention to minimize
`contamination.
`In order to remove contaminants such as nucleic acids,
`lipids, proteins, and
`microbes, a cleaning-in-place (CIP) step is often integrated in the purification protocol. Sodium
`hydroxide (NaOH)
`is probably the most extensively used cleaning agent
`for this purpose.
`Unfortunately, most protein-based affinity media show high fragility towards this extremely harsh
`environment, making themless attractive as resin-bound ligands. Asparagine has been shown to be
`the major contributor to the alkaline fragility. Here, this problem is addressed with a simple and
`straightforward strategy consisting in replacing asparagine residues with other amino acids.
`Applying this strategy,
`three different affinity ligands,
`important in large-scale production of
`different target molecules,
`i.e. antibodies and albumin, have been remarkably stabilized. These
`proteins include the albumin-binding domain (ABD) and the IgG-binding domain C2 of
`streptococcal protein G, as well as the IgG-binding B-domain analog Z ofstaphylococcal protein
`A. Multimerization of these stabilized domains has been performed in order to improve their
`performance as resin-bound ligands in chromatography. Additionally, stable linkers have been
`designed in order to not affect the function of the individual monomerunits. Also, it is shown that
`directed coupling using a C-terminal cysteine increases the capacity of the immobilized ligand
`compared to coupling using ordinary non-directed NHS-chemistry.
`
`In conclusion, such improvements presented in this thesis are attractive to obtain industrial
`implementation of protein-based affinity ligands, which normally show too high susceptibility to
`the alkaline conditions used during regeneration of the chromatography devices, or require to
`harsh elution conditions. Hence,
`these engineered proteins represent
`interesting ligands in
`purification of antibodies and albumin.
`
`linker
`Keywords: affinity chromatography, cleaning-in-place, deamidation, destabilization,
`engineering, protein engineering, stabilization, staphylococcal protein A, streptococcal protein G,
`turn/loop engineering
`
`© Susanne Giilich, 2002
`
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`Page 4
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`

`LIST OF PUBLICATIONS
`
`This thesis is based on the following publications, which will be referred to by
`their Roman numerals:
`
`I.
`
`Il.
`
`lil.
`
`IV.
`
`
`Giilich, $., Uhlén, M., Hober, S. 2000. Protein engineering of an
`IgG-binding domain allows milder elution conditions during affinity
`chromatography. J. Biotechnol. 76, 233-244.
`
`
`Giilich, S., Linhult, M., Nygren, P.-A., Uhlén M., Hober, S. 2000.
`Stability towards alkaline conditions can be engineered into a
`protein ligand. J. Biotechnol. 80, 169-178.
`
`
`Linhult, M., Gillich, S., Graslund, T., Nygren, P.-A., Hober, S. 2002.
`Linker engineering for
`improved performance of an affinity
`chromatographyligand. Manuscript.
`
`
`Giilich, S., Linhult, M., Stahl, S., Hober, $. 2002. Engineering
`streptococcal protein G for increased alkaline stability. Submitted.
`
`
`Linhult, M., Giilich, S., Grislund, T., Simon, A., Sjéberg, A., Nord,
`K., Hober, S. 2002. Stabilization of a staphylococcal protein A
`domain towards alkaline conditions. Manuscript.
`
`Page 5
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`

`

`TABLE OF CONTENTS
`
`INTRODUCTION
`
`1. Protein structure
`
`1.1. a-helix
`1.2. B-sheet
`1.3. Turn/loop
`1.4. Tertiary structure
`
`2. Chemical aging of proteins
`2.1. Modification of asparagine and glutamineresidues
`2.1.1. Parameters influencing deamidation
`2.2. Modification of aspartate and glutamate residues
`18
`2.3. Detection methods
`
`3. Protein engineering
`3.1. Characterization techniques
`
`4. Bacterial surface domains
`4.1. Staphylococcal protein A
`4.1.1. The Z-domain
`4.2. Streptococcal protein G
`4.2.1. The IgG-binding domains
`4.2.2. The albumin-binding domains
`
`5. Production and purification
`5.1. Affinity chromatography
`5.1.1. Matrix and immobilization strategies
`5.1.2. Ligand
`5.1.3. Capture and elution procedures
`5.1.4. Column regeneration
`5.1.5. Production and purification of IgG and albumin
`5.1.6. SPA and SPGaffinity chromatography
`
`PRESENT INVESTIGATION
`
`6. Destabilization of Z to allow milder elution conditions(1)
`6.1. Characterization and proof-of-concept
`
`7. Stabilization of ABD towardsalkaline conditions (II, HD
`7.1. Characterization and proof-of-concept
`
`8. Stabilization of C2 towards alkaline conditions (IV)
`8.1. Characterization
`
`9, Stabilization of Z towards alkaline conditions (V)
`9.1. Characterization
`
`10
`
`11
`11
`12
`
`13
`14
`16
`
`18
`
`19
`20
`
`21
`22
`25
`26
`27
`29
`
`30
`31
`32
`34
`35
`36
`
`37
`39
`
`41
`
`42
`44
`
`47
`50
`
`54
`55
`
`60
`61
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`

`10. Concluding remarks
`
`ACKNOWLEDGEMENTS
`
`ABBREVIATIONS
`
`REFERENCES
`
`65
`
`67
`
`68
`
`69
`
`Page 7
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`Page 8
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`INTRODUCTION
`
`1. Protein structure
`
`The building blocks in proteins and peptides are the amino acids. A total of 20
`
`different amino acids specified by the genetic three-letter code are known, capable
`
`of defining the enormous complexity of structures, stabilities, and functions
`
`associated with different proteins and peptides involved in widespread context in
`
`all biological systems. The amino acids are constituted of a central carbon atom
`
`that has attached a hydrogen atom, an amino group (NH2), a carboxyl group
`
`(COOH), and also a specific side chain unique for each amino acid and
`
`responsible for the different properties associated with a particular residue. Hence,
`
`each amino acid except glycine exhibits chirality and can exist in two different
`
`forms, L- and D-form. However, only the L-form is observed in proteins and
`
`peptides of biological origin. These amino acids span a reasonable range of
`
`variables such as size, shape, hydrophobicity, charge, polarity, and hydrogen-
`
`bonding capacity. The amino acids are linked to each other by peptide bonds
`
`forming relatively straight-line polymers,
`
`in which the carboxyl group of one
`
`residue is joined to an amino group of another residue. The angle of rotation
`
`around the central carbon and the carbonyl carbon is by convention denoted psi
`
`(y), and the rotation around the central carbon and the nitrogen is called phi (0).
`
`The relatively straight-line polymers are ordered into higher structural elements,
`
`i.e. secondary structures, including a-helix and B-sheet. Also, turns and loops can
`
`be included. The secondary structure elements are packed into structural motifs
`
`defining the tertiary structure. The secondary structure elements and the overall
`
`tertiary structure are stabilized by a numberof non-covalentstabilizing forces,1.e.
`
`hydrogen-bonds,
`
`ionic
`
`interactions, van der Waals
`
`interactions,
`
`and the
`
`hydrophobic effect. H-bonds are weak interactions but the cooperative effect,
`
`when a network of such interactions is composed, results in a very strong and
`
`specific interaction. The distance for a strong H-bond involving oxygen and
`nitrogen atoms is about 3 A between donor and acceptor. Van der Waals
`
`interactions are even weaker than H-bondsand are caused by transient dipoles in
`
`atoms. A protein can also be stabilized by covalent bonds formed between
`
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`

`

`cysteines or interactions with prosthetic groups including ions like iron, zinc,
`
`magnesium, and calcium.
`
`1.1. a-helix
`
`Pauling and colleagues described this structural element first (Pauling et al.,
`
`1951). Since then it has had a major influence on the understanding of protein
`
`structures. The most common a-helix consists of a right-handed coil, in which
`
`every backbone carbonyl group of residue n is hydrogen-bonded to a backbone
`
`NHgroupofresidue n+4, resulting in 3.6 residues per turn. Since the NH and CO
`
`groups have different polarities,
`
`the overall effect is a net dipole that gives a
`
`partial positive charge at the N-terminus andapartial negative charge at the C-
`
`terminus. Thus, there is a high preference for negatively charged residues near the
`
`N-terminus of helices and a high preference for positively charged residues near
`
`the C-terminus,
`
`that may interact with the helix dipole by simple electrostatic
`
`interactions (Chakrabartty et al., 1993). Attempts to increase the stability of a
`
`protein by introducing favorable negative charge at the N-terminus have been
`
`successfully performed (Nicholson et al., 1988). The ends of the helices can be
`
`called the N- and C-cap respectively, which may be defined as interface residues
`
`that are half inside and half outside of the helix. Additionally, the side chain of the
`
`N-cap residue can make a H-bond to the main chain atoms at the N-terminus
`
`(Chakrabartty et al., 1993). Asparagine is commonly occurring at
`
`the N-cap
`
`position due to its hydrogen-bonding property (Chakrabartty et al., 1993). Several
`
`studies have been performed in order to elucidate the preference of each residue
`
`for the helix structure. Ala, Leu, and Met have been reported to have high helix
`
`propensity, whereas Pro and Gly have low (O’Neil and DeGrado, 1990;
`
`Chakrabartty et al., 1991; Myers et al., 1997). Alanine is one of the most common
`
`residues in the helix interior (Chakrabartty et al., 1993). The special character of
`
`proline in general produces a bendinthestructure. Glycine is considered a helix
`
`destabilizer due to the conformational
`
`flexibility it
`
`introduces (O’Neil and
`
`DeGrado, 1990).
`
`10
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`

`

`1.2. B-sheet
`
`In contrast to a-helices, the B-sheets are composedofdifferent parts of the peptide
`
`chain. These different parts of the peptide chain are aligned either parallel or
`
`antiparallel to each other, resulting in B-sheets. All backbone carbonyl! groups are
`
`hydrogen-bonded to backbone NH in the adjacent strand. Different hydrogen-
`
`bonding patterns are observed for parallel and antiparallel alignment respectively.
`
`The different central carbon atoms are located alternated above and below the
`
`plane of the B-sheet, forming a “pleated” structure described by Pauling and
`
`Corey (1951). Moreover, the side chains point alternately above or below the
`
`plane thus allowing formation of hydrophobic and hydrophilic surfaces. The
`
`understanding of the interactions that determine the B-sheet stability is not very
`
`profound compared to a-helix. Aminoacids with high propensity for B-sheet have
`
`been reported to include Val, Ile, Thr, Phe, Tyr, and Trp, whereas Ala, Asp, Gly,
`
`and Pro are considered to exhibit low propensity (Minor and Kim, 1994a; Smith et
`
`al., 1994; Smith and Regan, 1995). However, it has been shownthatit is difficult
`
`to predict B-sheet propensity. The result very much depends on wherein a B-sheet
`
`the residue is situated in the model protein (Minor and Kim, 1994b). Formation of
`
`helical arrays of B-sheets in insoluble fibrils has been proposed to result in several
`
`protein misfolding diseases such as Alzheimer’s disease (Dalal and Regan, 2000;
`
`Ramirez-Alvaradoet al., 2000).
`
`1.3. Turn/loop
`
`Turns and loops are connecting secondary structure elements. While helix and
`
`sheet are considered as regular structures with repeating main chain torsion angles
`
`and arranged hydrogen-bonding,
`
`turns/loops do not exhibit these features.
`
`In
`
`general, turns are consisting of a few residues with specific backbone dihedral
`
`angles, whereas loops consist of less well-defined structures with varying lengths.
`
`Loops and turns are in general directed to the surface of the protein and therefore
`
`are rich in charged and polar residues (Leszczynski and Rose, 1986). Glycine
`
`residues have a strong preference for tight
`
`turns and loops in general. Also,
`
`proline is frequently observed in turn/loop sequences. These connecting segments
`
`are often implicated in function but when they are not intimately involved in a
`
`particular function, they can vary widely in both sequence and length without
`
`1]
`
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`

`

`affecting either the structure or the function (Leszczynski and Rose, 1986: Brunet
`
`et al., 1993; Castagnoli et al., 1994; Viguera and Serrano, 1997). Thus, some
`
`degree of
`
`tolerance
`
`exists
`
`for
`
`these
`
`structural
`
`segments. However,
`
`the
`
`thermodynamic stabilities and folding pathways can be affected by engineering
`
`these segments (Predki et al., 1996; Nagi and Regan, 1997; Nagi et al., 1999). By
`
`lengthening the loop, protein destabilization can be obtained due to higher energy
`
`requirements associated with the closure of longer loops compared to shorter ones
`
`(Nagi and Regan, 1997; Nagiet al., 1999).
`
`1.4. Tertiary structure
`
`In water-soluble proteins, secondary structure elements are packed together to
`
`form compact tertiary structures with a hydrophilic surface and a hydrophobic
`
`interior. Others, like membrane proteins, may be constructed otherwise. Different
`
`categories of structural groups exist including proteins madeup ofall a-helix,all
`
`B-sheet, and a mixture of these two elements,
`
`a/$8. ABD derived from
`
`streptococcal protein G (SPG) and Z derived from staphylococcal protein A
`
`(SPA) are examples of antiparallel
`
`three-helix bundles (Kraulis et al., 1996;
`
`Tashiro et al., 1997). The C2 domain of SPG exemplifies an o/B protein (Lian et
`
`al., 1992), and the CH2 domain of the Fc-fragment of IgG represents a domain
`
`constituted of B-sheet structure (Deisenhofer, 1981). Interestingly, the small-sized
`
`domains, ABD (paperII, III) and C2 (paper IV) of SPG, and Z of SPA (paper V),
`
`manage to form a very compact and stable framework probably because ofa well-
`
`defined hydrophobic core.
`
`The hydrophobic core is predominantly made up of hydrophobic residues, which
`
`seems to be one of the most critical aspects for stability of the folded state. The
`
`hydrophobic effect is considered to be one of the driving forces of the folding
`
`process, in whichthe protein proceeds from a high energy unfoldedstate to a low
`
`energy native state. Many different hypothetical models for the folding process
`
`have been proposed. Additionally, stabilizing interactions such as H-bonds, van
`
`der Waals interactions, salt bridges, disulfide bridges, and bound prosthetic
`
`groups assist proper native structure. The packing of residues appears to be
`
`extremely importantfor structure, stability, and function, and there is considerable
`
`complexity of assembling hydrophobic side chains into tightly packed cores,
`
`12
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`

`

`commonly including residues like Ala, Val, Ile, Leu, and Met (Munsonetal.,
`
`1996). Polar and charged residues might be seen but only if they can form
`
`satisfactory bonds. The core is quite closely packed but small changes in volume
`
`may be tolerated. Backbone movements or conformational changes in side chains
`
`may accommodate these small changes.
`
`The three-dimensional structure of a protein is determined by the amino acid
`
`sequence alone (Anfinsen, 1973). It has been proposed that a few key residues are
`
`defining the specific structure and the fold of the Bl domain of SPG has been
`
`transformed into that of ROP by changing just a few key residues in the Bl
`
`sequence (Dalal et al., 1997; Dalal and Regan, 2000). There might be non-local
`
`factors influencing the formation of secondary structures as shown byastudy, in
`
`which an 11-residue sequence could fold either into an a-helix or f-sheet
`
`depending on where in the B1 domain of SPG it was introduced (Minor and Kim,
`
`1996).
`
`2. Chemical aging of proteins
`
`Peptides and proteins are not chemically stable over time. This is especially the
`
`case in presence of water and small molecules, e.g. oxygen. A vast variety of
`
`different modifications
`
`are known that
`
`spontaneously occur under both
`
`physiological as well as non-physiological conditions. These include deamidation,
`
`isomerization and racemization. Oxidation,
`
`reduction, arginine conversion,
`
`hydrolysis, and B-elimination are also common modifications (Li et al., 1995;
`
`Reubsaet et al., 1998). These processes lead to covalent modifications that may
`
`severely alter the structure and hence,
`
`the function of the protein molecule.
`
`However, the position of the degraded residue in a protein determines the effect
`
`on the biological activity. Chemical modifications that significantly alter the
`
`structure in general
`
`lead to degradation of the protein. However,
`
`some
`
`modifications that do not degrade the protein tend to accumulate during the
`
`lifetime of a living organism.
`
`The spontaneous degradation of proteins due to deamidation of asparagines and
`
`glutamines is one of the most extensively studied protein modifications. The
`
`occurrence of asparagines and glutamines in proteins is widespread despite their
`
`obvious
`
`instability. Therefore,
`
`it has been proposed that
`
`the deamidation
`
`13
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`

`mechanism may play a general role in the aging of living organisms (Robinson
`
`and Robinson, 1991). In addition, a variety of essential processes may be timed by
`
`deamidation,
`
`including development and turnover of proteins (Robinson and
`
`Robinson, 200la). The reaction is under genetic control
`
`through sequence
`
`variation to adjust the degradationrate. If the reactions do not have anybiological
`
`purpose, asparagines and glutamines probably should have been evolutionary
`
`eliminated long ago since they truly are damaging (Robinson and Robinson,
`
`1991).
`
`2.1. Modification of asparagine and glutamineresidues
`
`Deamidation involves the amide side chains of asparagines and glutaminesthat in
`
`general are not very chemically reactive and do not ionize (Liu, 1992). However,
`
`they are polar and function as both H-bond donors and acceptors. The
`
`deamidation reaction is non-enzymatic and requires only water to occur (Geiger
`
`and Clarke, 1987). However, the reaction rate is dependent upon the concentration
`
`of hydroxide ions, and is therefore increased when the hydroxide concentration is
`
`increased. The proposed predominant pathway of deamidation of asparagines
`
`occurs via a succinimide intermediate. The reaction is an intramolecular
`
`cyclization,
`
`in which the deprotonated main chain peptide nitrogen on the C-
`
`terminal side, likely obtained by base catalysis, attacks the side chain carbonyl
`
`carbon (Geiger and Clarke, 1987) (Fig. 1). The succinimide intermediateis itself
`
`unstable in aqueous solution at neutral or alkaline pH and undergoes hydrolysis
`
`and racemization, which can take place at either of the carbonyl groups. As a
`
`consequence, the net product is a mixture of L- and D-aspartate and L- and D-
`
`isoaspartate (Geiger and Clarke, 1987). The ratio of iso-form to normal
`
`is
`
`approximately 3:1 at physiological conditions. In the pH interval 5-12 in several
`
`buffer systems,
`
`the deamidation appears to proceed entirely through this
`
`succinimide intermediate. However, at very low pH in the interval 1-2, a slow
`
`deamidation reaction is observed that
`
`is thought to use some other reaction
`
`mechanism, in which aspartic acid is the only product (Clarke et al., 1992). An
`
`alternative reaction mechanism to the succinimide mechanism has also been
`
`proposed by Wright (199 1a, b), in which a general acid catalyzes the reaction by
`
`protonating the side chain nitrogen of asparagine. A general base then attacks the
`
`14
`
`Page 14
`
`Page 14
`
`

`

`side chain carbonyl! carbon forming an oxyanion tetrahedral intermediate, which
`
`subsequently is transformed to aspartic acid. Since a charge difference arises
`
`when an amide residue is converted to a carboxylic acid residue, the reaction is
`
`readily detected by different techniques based on charge. As a consequence ofthe
`
`increased negative charge and the changed backbone configuration,
`
`the
`
`susceptibility to proteolytic degradation might
`
`increase due to opening of the
`
`protein structure.
`
`0 |c
`
`Asparagine
`
`N
`
`NH,
`
`Cc
`|
`Cc
`LN PBN YZ
`Cc
`Cc
`|
`|
`Oo
`R
`
`- NH
`
`0I
`
`|
`Oo
`
`oN Succinimid
`“.J
`
`N
`
`NZ
`
`uccinimide
`
`R
`
`—\“ Oo"
`Lo NONA
`
`Cc
`
`N
`
`|
`
`o
`
`R
`
`t
`
`(OS
`L\f?
`
`i
`
`|
`oO
`
`Aspartate
`
`1:3
`
`isoAspartate
`
`Fig. 1. Reaction mechanism for deamidation in peptides and proteins via
`
`formation of a succinimide intermediate.
`
`15
`
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`
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`
`

`

`Cleavage of the peptide bond at asparagine residuesis also a possible course of
`
`events. In contrast to the mechanism described for deamidation above, the side
`
`chain amide nitrogen attacks the C” thus, cleaves the peptide bond and forms a C-
`
`terminal succinimide. This reaction is thought to compete with the deamidation
`
`reaction. However, in general the deamidation reaction is faster (Tyler-Cross and
`
`Schirch, 1991),
`
`Glutamines may also experience the samereactions as asparagines but the product
`
`is glutamate instead. However,
`
`the rate in model peptides is generally much
`
`slower compared to asparagines (Robinson and Rudd, 1974). This is probably due
`
`to the resulting six-membered intermediate, which is not energetically favorable.
`
`Furthermore, the extra -CH> group imparts greater distance from the adjacent
`
`main chain nitrogen to the side chain carbonyl carbon of glutamine. An exception
`
`is the N-terminal glutamine that readily deamidates by cyclization with its own
`
`free N-terminus, resulting in pyrrolidone carboxylic acid. This is the only known
`
`case, in which glutamine deamidates faster than asparagines (Wright, 1991a,b).
`
`2.1.1. Parameters influencing deamidation
`
`It was early proposed that
`
`the deamidation rate was closely coupled to the
`
`sequence context (Robinson and Rudd, 1974). As a consequence, the rate can
`
`differ substantially in aqueous solutions at physiological pH between different
`
`peptides or proteins (Robinson and Rudd, 1974; Geiger and Clarke, 1987). Studies
`
`on peptides have unraveled that the most critical parameter is the nature of the
`
`residue on the C-terminal side of asparagine (Lura and Schirch, 1988). The
`
`preceding residue does not substantially affect the rate of deamidation. The Asn-
`
`Gly sequence is by far the most sensitive sequence (Geiger and Clarke, 1987;
`
`Robinson and Robinson, 2001a). The preponderance for glycine at unstable sites
`
`has been attributed to the unique character of the glycine residue, which lacks a
`
`side chain that can sterically interfere with the reaction mechanism. Therefore,
`
`glycine imparts exceptional flexibility to the peptide backbone. The dihedral
`
`angles psi (wy), which represents the rotation around the a-carbon and the peptide
`
`carbonyl carbon, and chil (x1), which represents the rotation around the a-carbon
`
`and the B-carbon, can approach optimal values for the mechanism whenglycineis
`succeeding. The presence of a C’ mayrestrict the range of motion thus disabling
`
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`

`the mechanism. Additionally, the electron-withdrawing or -donating effect of the
`
`C-terminal residue may influence the ease of deprotonation of the peptide bond
`
`nitrogen. The glycine side chain is lacking electron-donating substituents, which
`
`otherwise would decrease the deamidation rate. The Asn-Ser sequence is also
`
`highly unstable and even surpasses the Asn-Ala sequence even thoughalanine has
`
`a smaller side chain (Robinson and Robinson, 2001a). The fast rate observed for
`
`Asn-Ser maybe dueto facilitated deprotonation of the peptide bond nitrogen thus,
`
`enhancing the nucleophilicity. Another explanation might be that the hydroxyl
`
`group donates a H-bondto the side chain oxygen or nitrogen of asparagines thus,
`
`enhancing the electrophilicity of the carbonyl carbon atom (Kossiakoff, 1988).
`
`Hence, glycine, alanine, serine, and threonine result
`
`in increased deamidation
`
`rates when situated C-terminal to asparagine or glutamine. However, bulky and
`
`hydrophobic residues result in low deamidation rates (Robinson and Robinson,
`
`2001la).
`
`In the case with Asn-Pro the peptide bond nitrogen cannot be
`
`deprotonated and a succinimide intermediate cannot form (Geiger and Clark,
`
`1987).
`
`Prediction of potential deamidation sites is further complicated in proteins
`
`compared to peptides containing only a few residues. In proteins the secondary,
`
`tertiary, and quaternary structure must also be considered (Kossiakoff, 1988;
`
`Wearne and Creighton, 1989). The presence of Asn-Gly or Asn-Ser sequences
`
`does not necessarily indicate a site for deamidation. Sequencesin proteins with a
`
`well-defined three-dimensional structure that is not optimal for the deamidation
`
`mechanism show reduced deamidation rate in relation to the corresponding
`
`peptide. In somecasesit may bepossible that the fixed angles are optimal for the
`
`mechanism thus, the rate is increased in comparison to the corresponding peptide.
`
`However, in general short peptides showelevated deamidation rates compared to
`
`proteins (Geiger and Clarke, 1987). Hence, it may be very difficult to predict the
`
`sensitivity of a particular residue based on the amino acid sequencealone. For a
`
`proper investigation the three-dimensional structure as well as the flexibility of
`
`the polypeptide chain should be considered (Robinson and Robinson, 2001b). It
`
`mustalso be stressed that deamidation of one residue can initiate small changes in
`
`the tertiary structure creating a new configuration,
`
`that
`
`imposes a second
`
`deamidation at another site in the structure. Hydrogen-bonding may have a
`
`protective effect
`
`(Kossiakoff, 1988). Thus, a-helices may stabilize against
`
`17
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`

`

`deamidation
`
`due
`
`to
`
`conformational
`
`restrictions
`
`but
`
`also
`
`the
`
`reduced
`
`nucleophilicity of the backbone NH, which is H-bonded (Koskyetal., 1999; Xie
`
`and Schowen, 1999).
`
`The rate of deamidation is also dependent on other parameters like the
`
`temperature, buffer, pH, and ionic strength.
`
`Increased deamidation rates are
`
`observed when increasing temperature, pH, and ionic strength (Robinson and
`
`Rudd, 1974; Tyler-Cross and Schirch, 1991).
`
`2.2. Modification of aspartate and glutamate residues
`
`Since the modification of aspartates does not
`
`involve charge alterations the
`
`reaction is more difficult to detect compared to deamidation. This might result in
`
`possible hidden isomerization products when the function of the protein is not
`
`altered due to the modification. However, aspartates are prone to intramolecular
`
`succinimide formation similar
`
`to asparagines
`
`(Aswad et al., 2000). The
`
`isomerization and racemization reactions are suggested to involve the same
`
`mechanism as for deamidation. However, when comparing the reaction rates of
`
`aspartate containing peptides with the corresponding asparagine peptides,
`
`the
`
`succinimide formation is faster for the asparagine containing peptide (Geiger and
`
`Clarke, 1987; Stephenson and Clarke, 1989). At aqueous conditions the majority
`
`of the aspartates is in the deprotonated charged form presenting a poor leaving
`
`group to nucleophilic attack. The protonated uncharged form present at lower pH
`
`presents a much better leaving group (Stephenson and Clarke, 1989).
`
`Glutamate residues may undergo the same reactions. However,
`
`since the
`
`intermediate is a six-memberedring structure it is not energetically favorable. To
`
`my knowledge no reports on degradation of glutamate residues in peptides or
`
`proteins are available.
`
`2.3. Detection methods
`
`Since deamidation results in an increase of negative charge of the protein or
`
`peptide,
`
`this can readily be detected by changes in electrophoretic mobility.
`
`Isoelectric focusing (IEF) is therefore a sensitive and convenient method either
`
`under
`
`denaturing
`
`or
`
`non-denaturing
`
`conditions. Also,
`
`ion
`
`exchange
`
`chromatography (IEC) can be used to detect charge differences (Bischoff and
`
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`

`Kolbe,
`
`1994).
`
`Isoaspartyl
`
`residues
`
`can be detected by the
`
`isoaspartyl
`
`methyltransferase (PIMT). This is an enzymenaturally present in eukaryotic cells
`
`that specifically methylates L-isoaspartate residues. A drawback with this
`
`detection methodis that the enzyme does not recognize all L-isoaspartates with
`
`the sameaffinity due to substrate specificity for the n+] residue (Lowenson and
`
`Clarke, 1991). In addition,
`
`the residues must be located on the surface to be
`
`detected. The change in hydrophobicity and polarity can be analyzed by reversed-
`
`phase high performance
`
`liquid chromatography (RP-HPLC). Also, mass
`
`spectrometry can be used to detect protein modifications (Reubsaetet al., 1998).
`
`However, the 1 Da mass increase associated with deamidation might be difficult
`
`to detect. The sequence of the protein or peptide can also be decided by peptide
`
`sequencing.
`
`3. Protein engineering
`
`Protein engineering is a technique primarily used to create a novel protein that
`
`possesses an improved or novel property by changing one or several residues of
`
`an existing protein. Thus, protein engineering may be an attractive strategy to
`
`replace particular residues prone to modification and subsequent degradation,
`
`which is one of the scoops ofthis thesis. In addition, protein engineering has
`
`proven to be a valuable tool in determining the contribution of a particular amino
`
`acid to the enigmas of folding, stability, and function. The recent advances in
`
`biotechnology have resulted in extensive information on structure-function
`
`relationships and a palette of different genetic-engineering techniques
`
`is
`
`nowadays available. Strategies for both non-random mutagenesis and random
`
`mutagenesis have been developed and it
`
`is now routine work in laboratories
`
`worldwide. The specific properties of a protein can be altered in a non-random
`
`fashion by site-directed mutagenesis based on rational design. The geneis then
`
`modified in a predicted way through nucleotide substitutions,
`
`insertions, or
`
`deletions. Single
`
`amino acid substitutions
`
`can readily be produced by
`
`oligonucleotide-directed mutagenesis using the polymerase chain reaction (PCR)
`
`(Mullis and Faloona, 1987) with primers containing the modification. The primers
`
`can be designedto include the entire gene or a portion (cassette), and the PCR can
`
`be run in a one-step or a two-step manner (Higuchi et al., 1988). Another
`
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`

`

`approach is cassette mutagenesis,
`
`in which a part of the gene is cut out and
`
`replaced by a synthetic oligonucleotide. Combinatorial methods such as random
`
`mutagenesis and DNA shuffling produce libraries containing a vast number of
`
`different variants that can be analyzed simultaneously. Theselibraries can then be
`
`used to select for different functions and a particular protein can be s