peptide modification

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Peptide synthesis – chemistry and modifications
Peptides and proteins exhibit the largest structural and functional variation of all classes of biologically
active macromolecules. Biological functions as diverse as sexual maturation and reproduction,
enzyme inhibition, blood pressure regulation, glucose metabolism, thermal control, analgesia and
learning and memory are now thought to be regulated by peptides.
Peptide synthesis chemistry
Synthetic peptides are valuable tools in analysis of naturally occurring peptides or proteins. Since Emil
Fischer’s pioneering work in the early 1900’s, synthesis methods have improved continually –
especially with Merrifield’s development of solid-phase syntheses.
Besides the classical synthesis in solution, solid-support synthesis is now the most widely used
method to prepare synthetic peptides. The advantages of solid-support synthesis are its speed,
versatility, ease of automation and low costs.
However, peptide chemistry still remains a difficult and exacting science.
Solid-phase synthesis is usually carried out as follows:
1.
2.
3.
4.
5.
loading of C-terminal amino acid to resin (not shown below)
deprotection: removal of N-terminal protecting group (PG) at amino residue
activation of next amino acid at carboxy residue
coupling reaction
start synthesis cycle 2-4 again or cleave fully-synthesised peptide off resin
R
H
N
C
PG
H
H
R
C
N
OH
PG
activation
N
PG
C
C
O
C
O
H
deprotection
R
H
O
C
N
OH
H
R
H
O
H
C
H
O
etc.
C
O
coupling
H
N
PG
R
O
C
C
H
R
N
C
H
H
O
H
O
removal H
C
N
R
O
C
C
H
R
N
C
H
H
O
C
OH
1
Protecting groups (PGs)
One of the demanding parts in peptide synthesis is the necessity to block those functional groups that
must not participate in peptide bond formation. Such “protecting groups” are needed for all specific
side chain functions of DNA-encoded amino acids (p.e. the amino group of the amino acid that lends
its activated carboxyl group to the coupling reaction and for the carboxyl group of the amino acid that
will be acylated in its amine group).
In order to elongate the resulting dipeptide, one protecting group has to be removed. This has to be
done under such conditions that the peptide bond itself is not harmed and transient protecting groups
still stay on, too.
Solid phase synthesis requires two types of protecting groups:
a) Transient protecting groups for amino groups that form the peptide bond.
b) Permanent protecting groups for functional groups within the amino acid side chains. These
PGs have to be stable enough to sustain chemicals used to remove transient PGs.
Transient PGs for amino acids
Such PGs should be easily removable but still stable enough to survive the conditions of coupling
reactions and other manipulations. Two commonly used amino protecting groups are:
deprotection
t-Boc (t-Butoxycarbonyl)
under mild acidic conditions
with TFA (50% TFA in DCM)
advantage
stable towards catalytic hydrogenation,
can be used with Z group for side chain
protection
disadvantage final workup with HF necessary
Fmoc (9-fluorenylmethyloxycarbonyl)
mild basic, non-hydrolytic conditions
with primary or secondary amines:
20% piperidine in DMF
deprotection does not affect amide or t-butyl
protected side-chain esters,
stable towards tertiary amines
CH3
Fmoc
CH2
H3C
HF
O
tBoc
C
CH3
H3C
C
O
O
C
C
O
CH2
NH CH C
CH3
O
C
CH3
TFA
O
O
H
CH2 O
C
O
CH2
NH CH C
NH
NH
O
Piperidine
TFA
Permanent PGs for side chains
Since the different side chains of the DNA-encoded amino acids encompass the majority of the
common functional groups in organic chemistry, several different types of side chain protecting groups
are required for peptide synthesis.
These PGs are usually based on the Bzl or tBu group.
Amino acids with alcohols or carboxylic acids in the side chain can be protected either as Bzl ethers or
as Bzl or cHex esters. Another alternative is protection as tBu ethers or esters.
Other types of functional groups (amino group of Lys, thiol group of Cys, imidazole of His or guanidino
group of Arg) may require other special protecting groups.
Certain amino acids contain functional groups in their side chains that can cause unwanted side
reactions if not protected – but even with the semi-permanent protection, the situation is far from
satisfactory with certain amino acids.
2
In solid-phase synthesis this becomes even more acute, because acylating reagents have to be used
in excess to force the reaction to completion. The most commonly used protection strategies are the
tBoc/Bzl and Fmoc/tBu methods.
strategy
tBoc/Bzl
amino protection with
tBoc
side chain protection with Bzl or cHex,
either soft acid labile or
TFA stable and thallium/TFA labile or
soft acid stable
Fmoc/tBu
Fmoc
tBu,
ideally base stable and
weak acid labile
The following table displays a summary of special protection strategies for different amino acids:
tBoc/Bzl
Amino
Protecting
Comment
acid
group
Asn Gln unprotected
use of active ester or
DCC/HOBT or HBTU
Amino
acid
Arg
Asp Glu Bzl
Asn Gln
Ser, Thr
Tyr
Trp
Met
Lys
Arg
His
Trp
Met
Cys
Bzl
Bzl
2-Br.Z
2,6-di-Cl-Bzl
2-Cl-Z
Tos
Mts
Mbs
Tos
Bum
Bom
Trt
Dnp
unprotected
For
unprotected
Met(O)
tBu
MeBzl
Mob
Acm
Fmoc/tBu
Protecting
Comment
group
Mtr
Mtr requires long
Pmc
deprotection time
Pbf
Trt
removable TFA/TIS
Tmob
tBoc
unprotected
Asp Glu tBu
Ser, Thr, tBu
Tyr
TFA labile, protection
for one cycle
Lys
tBoc
stable to HF
requires thiolytic
cleavage prior to HF
His
Trt
Cys
Acm
StBu
Trt
removal by I2,
Hg(OAc)2
reduction with thiols
and phosphines
TFA/TIS
3
Coupling
For many years the most popular acylating agents have been carbodiimides. DCC (dicyclohexylcarbodiimide) mediated couplings have been used primarily in solid-phase synthesis.
The activation step of DCC-mediated coupling reactions is the formation of an O-acylisourea
intermediate of the carboxylic acid, which is favoured by nonpolar solvents, such ac DCM.
This intermediate can either
- react directly with resin-bound amines to form the desired amide
- or it reacts with further acids to yield the symmetric anhydride
- or it reacts with another agent, such as HOBt (1-hydroxybenzotriazole) to form a secondary
acylating reagent.
Certain side reactions of carbodiimides have led to the examination of other acylating agents. Two of
the more popular reagents are BOP and HBTU, both of which require activating bases.
The various preactivated tBoc or Fmoc amino acid species formed by these reagents are:
- symmetric anhydrides (carbodiimides, BOP, HBTU)
- acid chlorides (thionyl chloride)
- and esters.
For solid-phase synthesis, a large excess of acylating reagent (3-4 molar excess of resin-bound
amine) is recommended. Thus, under a normal and routine single coupling reaction, reaction rates of
>99% are usually observed. To ensure that the coupling reaction goes beyond that limit, a second
coupling reaction with a different solvent is used. Since DMF is a better solvent of peptide resins, a
st
mixed mode double-coupling procedure – i.e. symmetric anhydride in DCM (1 coupling) and
nd
DCC/HOBt or symmetric anhydride in DMF (2 coupling), can be useful for kinetics of coupling and for
accommodation to the varying solvating properties of the growing peptide chain.
Since a rapid coupling reaction is crucial for the Fmoc/tBu strategy to prevent cleavage side reactions
of Fmoc by resin-bound amines, coupling by symmetric anhydride in DMF is often chosen.
Some amino acids like His and Arg require special handling due to their proneness to certain side
reactions – as Fmoc amino acids are expensive and coupling reactions with symmetric anhydrides
have to be performed with excess reagents, active esters are chosen as alternatives.
Two useful active esters are HOBt and Odhbt (oxobenzotriazine) – either as preactivated forms or
generated in-situ.
4
Solid-phase peptide synthesis (SPPS)
SPPS consists of three distinct steps:
- chain assembly on a resin
- simultaneous or sequential cleavage and deprotection of the resin-bound, fully protected chain
- purification and characterisation of the target peptide.
Various chemical strategies exist for chain assembly and cleavage/deprotection methods, but
purification and characterisation are more or less invariant.
The two currently most popular SPPS strategies utilise either the acid labile tBoc or the base labile
Fmoc protecting groups. Each method involves fundamentally different amino acid side chain
protection as well as cleavage/deprotection.
The principles of stepwise solid-phase synthesis were first described by Merrifield:
- a tBoc amino acid (C-terminal amino acid of peptide) was covalently attached to an insoluble
polymeric support (the resin)
- the tBoc group was removed by TFA, the free amino terminus neutralised by TEA
- the next amino acid (with protected amino group) was activated and reacted with the resinbound amino acid to an amino-protected dipeptide
- DCM (dichloromethane) or DMF (dimethylformamide) served as primary solvent for
deprotection, coupling and washing
- Excess reagents and by-products were removed by simple filtration and washing.
rd
- the amino protecting group was removed and chain elongation continued with the 3 and
subsequent protected amino acids
- after the target-protected peptide had been built, all side chain protecting groups were
removed and the anchoring bond between peptide and resin was cleaved (HF or TFMSA)
Despite the use of optimised chemistry, not all peptides can be made with equal ease by SPPS. Some
amino acid sequences are more difficult to produce than others. In general, difficulties are rather
sequence-dependent than related to specific amino acid residues and they show a strong dependency
on solvents used for the coupling reaction.
Deprotection of peptides
Chemical synthesis of peptides – whether by solution or SPPS – requires a method to release the
desired peptide from its protecting groups. Acidolysis remains the most popular amongst the synthetic
strategies to cleave peptides from both resin and protecting groups.
Acidolytic deprotection is a major component in the overall scheme of SPPS approaches.
The first scheme – the tBoc/Bzl strategy uses the principle of differential acid lability, whereas the
second approach – the Fmoc/tBu strategy offers promising potential and a great selection for the acid
labile side chain protecting groups due to the wide range between basic and acidic cleavage.
Despite their usefulness for chemical synthesis of peptides, strong acids as deprotecting reagents
have led to many serious side reactions and to a loss of product invariably resulting from one common
origin: the modification of peptides by carbocations generated during the cleavage reaction.
Efforts have been made to reduce the carbocation formation during deprotection and to understand
the fundamental mechanism of non-aqueous acid cleavage reactions.
5
Purification of peptides
Purification strategies are usually based on a combination of separation methods which exploit the
physiochemical aspects of peptides or proteins, i.e.:
- their size
- their charge
- their hydrophobicity.
Among the various purification techniques can be found:
- size-exclusion chromatography
- IEC (ion-exchange chromatography)
- partition chromatography
- HPLC (high-performance liquid chromatography)
Thermo Electron uses RP-HPLC (reverse phase HPLC) as it is the most versatile and most widely
used HPLC method. It is called “reverse phase”, because it behaves in the opposite way to normal
phase chromatography:
- The stationary phase is silica – chemically bound with alkylsilyl compounds – resulting in a
non-polar, hydrophobic surface.
- Solute retention is mainly due to hydrophobic interactions between the solutes and the
hydrocarbonaceous stationary phase surface.
Polar mobile phases, usually water mixed with methanol, acetonitrile and/or other water
miscible organic solvents, are used for elution.
- Solutes are eluted in order of decreasing polarity (increasing hydrophobicity). Increasing the
polar component of the mobile phase increases retention of the solute.
Under identical HPLC conditions the retention of solutes increases proportionately with the carbon
chain length of bound groups (C18, C8, C4…HPLC columns). In general, the more polar (less
hydrophobic) compounds are best separated with mobile phases of lower organic content, as they are
more suited to the analysis of these compounds. The longer chain hydrocarbon phases interact best
with mobile phases of higher organic content and are, therefore, best suited to non-polar, hydrophobic
solutes.
6
Modifications of peptides
Peptides themselves can already inherit biological activity that can be observed in basic medical and
biological research. With the completion of HUGO its becoming more and more interesting to
characterise gene functions and the functions of their encoded proteins. Thus, researchers have
growing need for synthetic peptides – and depending on their experimental design, modified peptides
as well.
Among common standard modifications of peptides you can find:
- D-amino acids
- unnatural amino acids (6-Aminocaproic acid, Aminobutyric acid, Citrulline, Norleucine, etc.)
13
15
- Heavy amino acids (labelled with C and/or N)
- cyclisation
- phosphorylation or sulfurylation (at Ser, Tyr, Thr)
- biotinylation
- conjugation to carrier proteins (BSA, KLH, OVA)
- branching of peptides (MAPs – multiple antigenic peptides)
In general, there are some standard peptide moieties accessible to be modified:
- N-terminal amino group
NH2
- amino group of Lys
CH2
- thiol group of Cys
- hydroxyl groups of Ser, Thr, Tyr
CH2
- guanidine group of Arg
CH2
OH
- C-terminal carboxyl group
CH2 O
H
N
H
C
H
C
H
O
N
C
C
H
CH2
CH2 O
N
C
H
H
C
H
N
C
H
CH2
SH
O
C
OH
CH2
CH2
NH
C
HN
NH2
Amino group modifications (N-terminus or via Lys side chain)
All modifications carrying amine-reactive functional groups can be used.
Among the most commonly used ones, you can find:
- activated esters
- isothiocyanates
- carboxylic acids
Standard modifications that are coupled via amino groups are:
- biotin
- different dyes
- bifunctional linkers
- different acetylating groups
Thiol group modifications (via Cys side chain)
All modifications carrying thiol-reactive functional groups can be used.
Among the most commonly used ones, you can find:
- iodoacetamides
- maleimides
- alkyl halides
Standard modifications that are coupled via thiol groups are:
- different dyes
- KLH or BSA
7
Carboxyl group modification (C-terminus)
All modifications carrying carboxy-reactive functional groups can be used.
Among the most commonly used ones, you can find:
- amino groups
- amines
- bifunctional aminolinkers
Standard modifications that are coupled via carboxy groups are:
- amides
- chromophores
Single modifications – Amino acids
D-amino acids
Amino acids carrying four different groups on their α-C atom (i.e. asymmetric C atom, or C*) are chiral
substances. These α-amino acids can be found in respective L- and D-forms (enantiomers):
COOH
H2N
C
COOH
H
H
R
C
NH2
R
L-amino acid
D-amino acid
The predominant form in natural proteins is the L-form.
As some enzyme classes are enantioselective, i.e. they can distinguish between L- and D-forms and
specifically accept only one of the two forms as substrate, this enantioselectivity makes D-amino acids
a valuable tool in medicine (e.g. in peptide antibiotics) and enzyme assays.
Heavy Amino Acids
12
14
In contrast to standard amino acids composed of C and N atoms, heavy amino acids can be
13
15
substituted with C and/or N atoms. These heavy amino acids are non-radioactive, but 1 Da heavier
than the standard amino acids.
This molecular weight difference makes them useful tools for quantitative analysis of peptides by Mass
Spectrometry (MS) and Nucleic Magnetic Resonance Spectroscopy (NMR) e.g. for determination of
protein structure and dynamics.
Among Thermo’s standard heavy amino acids you can find:
amino acid (aa) heavy isotope
Gly
L-Ala
L-Leu
L-Phe
L-Pro
L-Val
13
15
U- C2, 98%; N, 95%
13
C, 99%
15
N, 98%
13
15
U- C3, 98%; N, 95%
13
C, 99%
15
N, 98%
13
15
U- C6, 98%; N, 95%
13
C, 99%
15
N, 98%
13
15
U- C9, 98%; N, 95%
13
C, 99%
15
N, 98%
13
15
U- C5, 98%; N, 95%
15
N, 98%
13
15
U- C5, 98%; N, 95%
13
C, 99%
15
N, 98%
difference to
standard aa
+ 3 Da
+ 1 Da
+ 1 Da
+ 4 Da
+ 1 Da
+ 1 Da
+ 7 Da
+ 1 Da
+ 1 Da
+ 10 Da
+ 1 Da
+ 1 Da
+ 6 Da
+ 1 Da
+ 6 Da
+ 1 Da
+ 1 Da
8
Unnatural amino acids, special amino acids and protecting groups
In contrast to the 20 natural amino acids (or proteinogenic), these amino acids are not encoded by the
Universal Genetic Code – usually they can be found in nature as metabolic products, especially in
plants and bacteria.
Some examples can be found below:
O
H
H
N
C
NH2
NH2
CH2
CH2
CH2
CH2
CH2
CH2
C
NH2
H
COOH
C
NH2
COOH
Citrulline
Ornithine
Thermo Electron’s range of special amino acids comprises the following molecules. Please inquire if
you do not find your desired amino acid listed.
ε-Acetyl-Lysine
COOH
H2N
CH
CH2
CH2
CH2
CH2
CH3CO
NH
β-Alanine (3-amino-propionic acid)
is the only natural occurring β-amino acid, present e.g in panthothenic acid.
COOH
CH2
H2N
CH2
Aminobenzoic acid
COOH
NH2
6-Aminocaproic acid (Aca, 6-Aminohexanoic acid)
This amino acid is often used as a linker to increase the distance between the peptide and an
additional modification, e.g. a fluorescent dye.
COOH
CH2
CH2
CH2
CH2
H2N
CH2
9
Aminobutyric acid (Abu)
γ-aminobutyric acid (or GABA) is an inhibitory transmitter of the central nerval system. It enhances
permeability of postsynaptic membranes for chloride ions, thus leading to hyperpolarisation and
consequently to an increase of the membrane’s activation potential.
COOH
CH2
H2N
CH
CH3
Citrulline
is a metabolic reagent in the urea metabolism pathway of many terrestric vertebrates. In this pathway,
unwanted ammonia is being detoxified and eliminated.
O
H
N
C
NH2
CH2
CH2
CH2
H
C
NH2
COOH
Cysteine, Acm (Acetamidomethyl) protected
this specially protected Cys is used to selectively form disulfide bridges.
COOH
H2N
C
H
CH2
S
CH2
NH
C
O
CH3
Dimethyl-Lysine
COOH
H2N
C
H
CH2
CH2
CH2
CH2
N
H3C
CH3
Hydroxy-Proline (Hyp)
is present almost exclusively in structural proteins (e.g. collagens or connective tissues in plant cell
walls or mammals). It is formed during a posttranslational modification of proline in cells.
COOH
CH
HC
HC
NH
CH2
OH
10
Mercaptopropionic acid
COOH
CH2
CH2
SH
Methyl-Lysine
COOH
H2N
C
H
CH2
CH2
CH2
CH2
N
H
CH3
3-Nitro-Tyrosine
COOH
H2N
C
H
CH2
NO2
OH
Norleucine (Nle = 2-amino hexanoic acid)
COOH
H2N
CH
CH2
CH2
CH2
CH3
pyro-Glutamic acid (Pyr)
is a common N-terminal amino acid modification in many biologically active peptides (hormones).
COOH
NH
O
Z (Carbobenzoxyl)
special protecting group for N-terminus.
C
O
O
11
Single Modifications - standard modifications
Biotin
Biotin (or vitamin H) is a small biologically active molecule with a molecular weight of 244,31 Da.
It acts as a co-enzyme in living cells. With its highly specific affinity towards streptavidin, it is used in
various biotechnology assays for quality and quantity testing.
O
HN
NH
OH
S
O
Farnesyl
is a potential substrate to study demethylase activity in enzyme assays.
CH3
CH3
H3C
CH3
Formic acid (Formyl)
O
C
H
OH
Myristic acid (Myristoyl)
COOH
Palmitic acid (Palmitoyl)
COOH
Phosphorylation
of Ser, Thr and Tyr is one of the more common modifications of amino acids in nature.
Many hormones can adapt the activity of specific enzymes by increasing their phosphorylation state of
Ser or Thr residues. Growth factors (like insulin) can trigger phosphorylation of Tyr.
The phosphate groups on these amino acids can be quickly removed, thus Ser, Thr and Tyr function
as molecular switches during regulation of cellular processes (e.g. cancer proliferation).
COOH
H2N
C
H
CH2
OPO3-
COOH
COOH
H2N
-
C
H
C
H
CH2
CH
O3PO
H2N
CH3
OPO3-
Stearic acid (Stearyl)
COOH
12
Succinic acid (Succinyl)
COOH
CH2
CH2
COOH
Sulfurylation
at Ser, Thr and Tyr is another modifcation of amino acids in nature.
Activity of many enzymes depends on the oxidation state of SH-groups in these residues.
H2N
H2N
CH
CH2
OSO3
-
COOH
COOH
COOH
O3SO
H2N
CH
CH
CH2
CH
CH3
OSO3-
13
Single Modifications - dyes and quenchers
AMC (7-Amino-4-methyl-coumarin)
UV-excitable dye, used in enzyme assays using cuvettes or flow cytometry.
H2N
O
O
COOH
CH3
Dye
Excitation maximum
Emission maximum
AMC
353 nm
422 nm
Molar Extinction
Coefficient
19.000
Cy3, Cy5
are dyes with extremely high extinction coefficients and fluorescence. Thus, they are especially
suitable for very sensitive localisation assays of peptides in cells.
Their disadvantage is the high instability of the molecules under peptide synthesis conditions.
Therefore the yields are comparatively low.
+
N
N
HO
N
OH
+
N
HO
OH
Molar Extinction
Coefficient
150.000
250.000
Dye
Excitation maximum
Emission maximum
Cy™3
Cy™5
550 nm
650 nm
570 nm
670 nm
Dabcyl
Dabcyl is a non-fluorescent dye predominantly used as a quencher for other fluorophores (esp.
Fluorescein type dyes, EDANS..).
If Dabcyl is coupled to a peptide in close proximity to a fluorophore, it absorbs the emitted light of the
fluorophore. Enlarging this distance (i.e. by enzymatic cleavage of the peptide) results in excitation of
the fluorophore with an emission signal that can be detected.
N
(Me)2N
OH
N
O
Excitation maximum Emission maximum Molar Extinction
Coefficient
Dabcyl
453 nm
none
32.000
Dye
14
Dansyl
Dansyl is also used as a fluorophore quencher. Unlike Dabcyl, it inherits own fluorescence and thus
might not be as useful for highly sensitive assays.
H3C
CH3
N
SO3H
Excitation maximum Emission maximum Molar Extinction
Coefficient
Dansyl
335 nm
526 nm
4.600
Dye
2,4-Dinitrophenyl (DNP)
is a non-fluorescent dye that can be used as a fluorophore quencher (see Dabcyl for more details).
O2N
NO2
Dye Excitation maximum Emission maximum Molar Extinction
Coefficient
DNP
348 nm
none
18.000
DNP-Lysine
is a non-fluorescent dye that can be used as a fluorophore quencher (see Dabcyl for more details).
COOH
H2N
C
H
CH2
CH2
CH2
CH2
N
H
O2N
NO2
Dye
Excitation maximum Emission maximum Molar Extinction
Coefficient
DNP-Lysine
348 nm
none
18.000
15
EDANS (5-((2-aminoethyl)amino)napthalene-1-sulfonic acid)
a commonly used dye in FRET (fluorescence resonance energy transfer) peptides in combination with
Dabcyl as quencher.
NH2
CH2
H
CH2
N
SO3
Dye
Excitation maximum Emission maximum Molar Extinction
Coefficient
EDANS
335 nm
493 nm
5.900
Fluorescein
the commonly used fluorescent dye in confocal laser-scanning microscopy and flow cytometry
applications.
HO
O
O
COOH
HOOC
Dye
Excitation maximum
Emission maximum
Fluorescein
495 nm
520 nm
Molar Extinction
Coefficient
83.000
NBD (7-nitrobenz-2-oxa-1, 3-diazole)
a fluorescent dye, used for amine modification.
N
O
N
NO2
Dye Excitation maximum Emission maximum Molar Extinction
Coefficient
NBD
486 nm
543 nm
27.000
p-Nitro-Aniline
a chromogen used as colorimetric enzyme substrate in many standard enzyme assays in cuvettes.
NH2
NO2
Dye
Excitation maximum Emission maximum
p-Nitro-Aniline
410 nm
none
16
Rhodamine B
represents one among a numerous range of rhodamine dyes, used in fluorescence assays.
C2H5
C2H5
N
O
N
H5C2
C2H5
COOH
HOOC
Dye
Excitation maximum Emission maximum Molar Extinction
Coefficient
Rhodamine B
550 nm
580 nm
90.000
Tamra
the most commonly used rhodamine dye in fluorescence assays.
CH3
CH3
N
+
O
N
H3C
CH3
COOH
HOOC
Dye
Excitation maximum
Emission maximum
Tamra
544 nm
576 nm
Molar Extinction
Coefficient
90.000
17
Single modifications – modifications for antibody production
KLH, BSA, OVA Conjugates
Peptide-protein conjugates are used for antibody production against peptides. Peptides alone are
mostly too small to elicit a sufficient immune response, so carrier proteins containing many epitopes
help to stimulate T-helper cells, which help induce the B-cell response.
It is important to remember that the immune system reacts to the peptide-protein conjugate as a whole
– so there will always be a portion of antibodies to the peptide, the linker and the carrier protein.
Among the most common carrier proteins one can find:
•
KLH (Keyhole Limpet Hemocyanin), a copper containing, non-heme protein found in
5
arthropods and mollusca. It is isolated from Megathura crenulata and has a MW of 4.5 x 10 ~
7
1.3 x 10 Da. KLH is the most commonly selected carrier due to its higher immunogenicity
compared to BSA.
•
BSA (Bovine Serum Albumin), a plasma protein in cattle, belonging to the most stable and
3
soluble albumins. It has a MW of 67 x 10 Da – containing 59 lysines. About 30-35 of these
primary amines are accessible for linker conjugation, which makes BSA a popular carrier
protein for weak antigenic compounds.
A disadvantage of BSA is, that it is used in many experiments as a blocking buffer reagent. If
antisera against peptide-BSA conjugates are used in such assays, false positives can occur,
because these sera also contain antibodies to BSA.
•
OVA (Ovalbumin), a protein isolated from hen egg whites, with a MW of 45 x 10 Da.
It is a good choice as second carrier protein to verify if antibodies are specific for the peptide
alone and not the carrier protein (e.g. BSA)
3
MAPs (Multiple Antigen Peptides)
are branched peptides that can be used for direct immunisation to produce antibodies. MAPs are
usually “big enough” to raise the immune response.
The antigenic peptide of interest is being synthesised directly on the branched MAP structure.
MAPs are available as MAP 4 (4 branches) or MAP 8 (8 branches) molecules:
Schematic graph of a MAP 4:
antigenic peptide
antigenic peptide
antigenic peptide
antigenic peptide
18
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