Download OMNI kit - EnzyPep

The OMNI kit is the ultimate system for
synthesizing long peptides. It utilizes an
enzyme to efficiently couple peptide
fragments of various lengths to
assemble your desired product.
OMNI kit
peptide synthesis kit
EnzyPep B.V.
Contents
OMNI Kit Components .................................................................................................................................. 2
Storage .......................................................................................................................................................... 2
Introduction .................................................................................................................................................. 3
Advantages of the OMNI Kit: ........................................................................................................................ 5
Description: ................................................................................................................................................... 5
Summarized Substrate Profile for OMNI Reaction: .................................................................................. 8
Substrates ................................................................................................................................................. 9
Reaction .................................................................................................................................................. 10
Recommended Procedure ...................................................................................................................... 11
Additional Materials Needed ...................................................................................................................... 13
OMNI Reaction Guidelines .......................................................................................................................... 14
Troubleshooting .......................................................................................................................................... 15
CEPS information ........................................................................................................................................ 16
Other Products - Specific Ligases ................................................................................................................ 16
Ordering Information .................................................................................................................................. 16
Appendix I ................................................................................................................................................... 17
Compatible Reagents .............................................................................................................................. 17
Incompatible Reagents ........................................................................................................................... 17
References: ................................................................................................................................................. 18
Contact us ................................................................................................................................................... 19
Notice to Purchaser ................................................................................................................................ 19
Certificate of Analysis .................................................................................... Error! Bookmark not defined.
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OMNI Kit components
Omniligase-1 stock solution:
Omniligase-1 in 25 mM Tricine buffer, pH 7.5 (0.33 mg/mL) …….…………………….334 µL
Coupling reaction stock solution:
Acyl donor (Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 10 µM) and nucleophile (H-Ala-LeuArg-NH2, 15 µM) in water………………………………………………………………….50 µL
Storage
Store at -20°C. Avoid freezing re-thawing steps.
Recommendation: Aliquot (e.g. 5 µL) the omniligase-1 enzyme immediately and store at -20°C.
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Introduction
Chemo-enzymatic peptide synthesis (CEPS), the enzymatic ligation of chemically
synthesized peptide fragments, is potentially one of the most cost-efficient technologies for the
synthesis of medium-sized and long peptides (e.g. 20-100 amino acids) and peptide-to-protein
couplings. When compared to chemical fragment coupling, CEPS offers several notable
advantages: protection of the side-chain functionalities is not necessary, racemization is absent,
and it uses less solvents. The OMNI kit is the ultimate system for assembly of long peptides and
peptide-to-protein couplings. It utilizes a peptide ligating enzyme to efficiently couple peptide
fragments of various lengths into your desired product.
The coupling enzyme peptiligase1 was developed by EnzyPep B.V. (Geleen, the Netherlands) for
application in the chemo-enzymatic synthesis of peptides (CEPS). Omniligase-1 is a fourth
generation peptiligase variant that has been selected for its exceptionally broad substrate
specificity.
Typically, omniligase-1 catalyzes the reaction between an ester (acyl donor) and amine (acyl
acceptor, nucleophile) fragment (Figures 1, 2). The enzyme can be used to perform linear peptide
synthesis, but also catalyzes peptide macrocyclization very efficiently (Figure 1B).
Figure 1: Schematic illustration of the OMNI reaction. Panel A: Linear peptide coupling
reaction. Panel B: Head-to-tail peptide cyclization.
A
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B
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Advantages of the OMNI Kit:
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Fast peptide-peptide, peptide-to-protein coupling reactions within minutes, under mild
aqueous conditions.
No racemization.
Protection of substrate side residues is not required.
Broad substrate scope ligase, not needing recognition tags, results in traceless coupling.
Although intrinsically traceless, the use of peptide linkers between peptides, peptide
fragments or proteins is possible (e.g. for conjugation).
Simple, efficient peptide cyclization.
Description:
Enzyme
Omniligase-1 is a highly engineered ligase developed by EnzyPep BV. It combines the
properties of the hyperstable, cation-independent subtilisin BPN’ variant Sbt1492 and of
thiolsubtilisins, such as subtiligase3. The enzyme is exceptionally robust and displays activity in
the presence of organic co-solvents (e.g. up to 50% DMF or 50% DMSO), chaotropic agents (up
to 4M GnCl, 2M urea), disrupting agents (SDS, EDTA, Triton X-100) and reducing agents (DTT
and TCEP). It is stable at temperatures up to 55°C (unfolds at 70°C).
Substrate scope of omniligase-1. Similar to peptiligaseError! Reference source not found., omniligase-1
has six substrate recognition subsites; four at the acyl donor binding site; S1-S4, and two at the
amine site; S1' and S2' (Figures 2, 3; nomenclature of Schechter & Berger). The full substrate
scope of the enzyme has been determined (Figure 3). The substrate scope of omniligase-1 is very
broad.
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Figure 2. Substrate recognition sites of omniligase-1. S1-S4 are substrate pockets at the acyl
donor site. At the acyl acceptor side, S1'and S2' substrate binding pockets are present.
S4
S3
S2
S1
Y-(Z)m-P4P3P2P1-Ester +
Acyl donor (ester) fragment
S1'
S2'
H2N-P1'P2'-(Z)n-OH (or -NH2 or Y)
Nucleophilic (amine) fragment
where the fragments are water-soluble, and
Z = unprotected or protected amino acid or non-peptidic moiety
Y= terminal protecting group (optional)
P4 = hydrophobic L-amino acid or protected L-amino acid
P3 = any L-amino acid
P2 = any L-amino acid except Pro (preferably not Glu, Asp and Trp)
P1 = any L-amino acid except Pro (preferably not Gly and Asp)
P1' and P2‘= any L-amino acid except Pro
m, n = any number
Apart from P4-P1, P1' and P2', all other components (X,Y) can be of nonpeptidic nature, e.g. polymer
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Figure 3. The substrate profile of omniligase-1.
The acyl donor pockets S4-S1 determine the reaction rate (product yield after 15 minutes is
depicted above, maximum conversion after 60 min or longer), the amine pockets S1' and S2'
determine the reaction yield. The S4 pocket prefers hydrophobic P4 amino acids. If appropriate,
hydrophobicity of P4 can be achieved by using side-chain protection. The P1-P4, P1' and P2'
pocket preference are depicted in Figure 3.
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Omniligase-1 accepts all amino acids except proline in the S1'and S2' substrate binding pockets
(Figure 3).
Summarized substrate profile for OMNI reaction:
Y-(Z)m-P4P3P2P1-Ester +
Acyl donor (ester) fragment
H2N-P1'P2‘-(Z)n-OH (or -NH2 or Y)
Nucleophilic (amine) fragment
where the fragments are water-soluble, and
Z = amino acid
Y= terminal protecting group (optional)
P4 = hydrophobic L-amino acid or protected L-amino acid
P3 = any L-amino acid
P2 = any L-amino acid except Pro (preferably not Glu, Asp and Trp)
P1 = any L-amino acid except Pro (preferably not Gly and Asp)
P1' and P2‘= any L-amino acid except Pro
m, n = any number
Apart from P4-P1, P1' and P2', all other components (X,Y) can be of nonpeptidic nature, e.g. polymer
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Substrates
Acyl donor
Alkyl, aryl and thioesters can be utilized, but carboxyamidomethyl (Cam) esters or substituted
Cam esters (e.g. –Cam-Leu-OH or –Cam-Leu-NH2) are recommended. The substitution of the
Cam ester can be used to alter the polarity, and thus the solubility of the ester fragment. Cam
esters and substituted Cam esters can be generated on resins commonly used for solid phase
peptide synthesis4.
Because omniligase-1 has such a broad specificity, the acyl donor (ester) fragment normally
requires N-terminal protection to prevent formation of cyclic peptides or polymers. The use of a
phenylacetyl protection group is recommended; it can be removed easily with commercially
available acylases (e.g. immobilized PenG acylase from Iris Biotech EZ60040).
Acyl acceptor
The nucleophilic (amine) peptide fragment requires no protection at all. All amino acids except
prolines in the P1' and P2' are accepted.
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Reaction
The reaction is exceptionally fast (typically 5-10 min at an enzyme:product ratio of 1:100 for a 2fragment coupling). For convenience, most coupling reactions can be performed at ambient
temperature. Conversion is usually above 80% (hydrolysis ≤ 20%) at a 1.1 to 1.5 molar excess of
the amine fragment. Conversion generally improves with the length of the fragments. Details can
be found in the following section.
Important considerations:
Substrate concentration
The amine fragment concentration is crucial for the reaction yield. A higher concentration
correlates to a better coupling yield.
pH
A pH between pH 8.0 - 8.5 is optimal and required. At lower pH, the amine fragment is
protonated, and thus unreactive; at higher pH, the Cam-ester is chemically hydrolyzed. Because
peptides are usually acidic after TFA (trifluoroacetic acid) cleavage and a preparative HPLC
step, it is critical to check the pH (and adjust accordingly) before starting the enzymatic coupling
reaction.
Temperature
A temperature range of 4 to 50 °C can be used, however ambient temperature is preferred.
Additives
Do not use any reagents that react with thiols such as maleimides or iodoacetamides, since they
will inhibit the active site cysteine.
Head-to-tail cyclizations
The use of omniligase-1 with an unprotected ester fragment can be employed to manufacture the
corresponding head-to-tail macrocyclic peptide without significant generation of dimers or
polymers.
Because of the broad substrate specificity, most macrocycles offer several coupling options with
no restriction to Gly or Pro in order to prevent racemization.
The reaction is intrinsically traceless, but can also be used with peptide linkers between other
peptides, peptide fragments or proteins.
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Recommended Procedure
Omniligase-1 is provided with a test reaction kit containing:
-334 µL omniligase-1 solution at a concentration of 330 µg/mL
-55 µL peptide test coupling solution (pH 2.5) containing 10 mM Ac-Asp-Phe-Ser-Lys-LeuOCam-Leu-OH (Ac-DFSKL-OCam-L-OH) and 15 mM H-Ala-Leu-Arg-NH2 (H-ALR-NH2) in
MilliQ water.
These solutions should be stored at -20°C. To perform the omniligase-1 test reaction, please
follow the protocol below:
Before starting:
5 glass HPLC vials
1. Prepare fresh 1M pH 8.5 potassium phosphate buffer (Solution A).
2. Prepare a TCEP stock solution (100 mg/mL) in MilliQ water; this can be stored at -20°C
(Solution B).
3. Prepare a quenching solution of water/acetonitrile/methanesulfonic acid (48/48/4, v/v/v) to
stop the enzymatic reaction (Solution C).
Reaction:
1. Thaw the frozen acyl donor and amine fragment stock solution as well as omniligase-1 stock
solution (it is advised to immediately make aliquots and store at -20°C, e.g. of 5 µL). Keep
working solution on ice.
2. Add 475 µL quenching solution (Solution B) to four of the HPLC vials (1, 2, 3 and 4)
3. Add 75 µL phosphate buffer (Solution A) to the fifth, empty “reaction” HPLC vial. Add 2.5
µL TCEP (Solution B) and 2.0 µL omniligase-1 solution to the reaction vial.
4. Add 25 µL of the peptide Cam-ester and amine stock solution to the reaction vial, mix well
and adjust pH if required1 (final pH ~ 8.0 to 8.5).
5. Monitor the reaction over time. After 1, 15, 30 and 60 minutes quench the reaction by adding
25 µL of the reaction mixture to the quenching HLPC vials 1, 2, 3 and 4, respectively.
6. Analyze the samples by HPLC or LC-MS. See below an overlay of the different HPLC spectra
and corresponding masses.
1
It is critical to measure and adjust the pH value for the omniligase reaction to reach high conversions and yields.
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Figure 5. Reaction profile monitored in time.
Product (M = 989.6)
Ac-DFSKLALR-NH2
Amine (M = 357.3)
H-ALR-NH2
Hydrolyzed ester (M = 650.3)
Ac-DFSKL-OH
Ester (M = 821.4)
Ac-DFSKL-OCam-L-OH
Analysis Conditions
Analytical HPLC was performed on an Agilent 1260 Liquid Chromatograph, using a reversedphase column (Dr. Maisch, C18, 5 µm particle size, 250 × 4.6 mm) at 40°C. UV detection was
performed at 220 nm using a UV-VIS 204 Linear spectrometer. The gradient program was: 0-12
min linear gradient ramp from 30% to 50% eluent B (eluent A: 0.5 mL/L methane sulfonic acid
in H2O; eluent B: 0.5 mL/L MSA in acetonitrile). The flow rate was 1 mL/min and injection
volumes were 25 μL.
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Additional Materials Needed
Materials:
Glass HPLC vials
Solutions:
Solution A: 1M K-phosphate buffer, pH 8.5
Solution B: TCEP [tris-(2-carboxyethyl)phosphine]stock solution (100 mg/mL) in MilliQ water
Solution C: MilliQ water/acetonitrile/methanesulfonic acid (48/48/4, v/v/v)
1-5 M NaOH or HCl, for pH adjustments
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OMNI Reaction Guidelines
Identification of ideal coupling positions
To identify suitable fragments for coupling, find a coupling position with a hydrophobic P4
residue and favorable amino acids in P1' and P2' pockets. Please note that a combination of suboptimal amino acids can lead to a low yield.
There should be no prolines near the coupling site.
High yield and conversion
Use as high a concentration of the amine fragment as possible.
Always check (and adjust) the pH before starting the reaction.
Addition of TCEP (or optionally DTT) is preferred as it activates the enzyme; oxidative
conditions, disulfides or maleimides can inhibit the enzyme.
Analysis
Analyze the coupling reaction by HPLC-MS, use acidic conditions (<pH 3, see above) to quench
the reaction.
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Troubleshooting
(Almost) no conversion at all:
Check the enzyme activity and sequence of acyl donor fragment (is the enzyme still frozen or
been thawed too many times?).
Are all substrates in solution? Precipitated/aggregated peptides can inhibit the enzyme.
Are reductive conditions being used? (oxidative conditions can inhibit omniligase-1)
Only (or excessive) hydrolysis:
Is it possible to increase the amine concentration?
Check pH.
Are the two amino acids at the P1'and P2' positions sub-optimal? Try to choose a different
coupling position.
Are the two amino acids at the P1' and P2' pocket positions accessible (e.g. when coupling to
specific proteins)? Try using disrupting agents (urea, GnCl) or a peptide spacer.
If none of the above works, or you are in need of an enzyme for a specific coupling reaction,
please contact EnzyPep (contact details at last page).
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CEPS information
Peptiligases are the patented and proprietary products of EnzyPep (Brightlands Campus, 6167
RD Geleen, Netherlands).
Omniligase-1 is available under license on request for using CEPS technology to manufacture
pharmaceutical grade peptides and peptide conjugates.
Other products-Restriction ligases
Other peptiligases are available in both research and pharmaceutical grades. At the present time,
over 100,000 unique peptiligases can be expressed, of which about 250 are well-characterized.
In contrast to omniligase-1, many other peptiligases show very tight specificity, only permitting
the CEPS coupling of closely defined sequences. This tight specificity
 avoids the necessity of N-terminal protection of the ester fragment,
 assists purification by not coupling fragments with false amino acids in the P4-P1, P1' and
P2' positions,
 allows chain-specific coupling to heterodipeptides and branched chain peptides.
EnzyPep also offers feasibility study services to help identify (a) whether CEPS can be used to
assemble a particular polypeptide, and (b) provide or design peptiligases to optimize a CEPS
procedure.
If you are interested in any of these additional products or services, please contact EnzyPep at
[email protected] or tel: +31 (0)46 47 60 675
Ordering information
Please contact IRIS Biotech GmbH. http://www.iris-biotech.de/
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Appendix I
Compatible reagents
The table below shows the maximum concentration of each reagent tested at EnzyPep. Higher
levels may be acceptable, but they should be tested before use. Please note that some of these
reagents may partially or completely denature omniligase-1.
Reagent
DTT (dithiothreitol)
TCEP tris-(2-carboxyethyl)phosphine
EDTA
SDS
Triton X-100
Urea
Guanidinium chloride
Acceptable Concentration
1-5 mg/mL; 6.5-30 mM
1-5 mg/mL; 3.5-17.5 mM
10 mM
5% (w/v)
5% (w/v)
6M
2M
Incompatible reagents
These reagent are incompatible with the ligation reaction.
Reagent
Maleimide
Iodoacetamide
Epoxides
High concentrations of alcohols (transesterification)
High concentrations of thiols (thioester synthesis)
Ammonia or other primary amines (amide synthesis)
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References:
1. Toplak, A., Wu, B., Nuijens, T., Quaedflieg, P.L.J.M. & Janssen, D.B. (2016) Adv. Synth.
Catal. (in press).
2. Jackson, D. Y., Burnier, J., Quan, C., Stanley, M.;Tom, J., Wells, J. A. (1994) Science, 266,
243.
3. Braisted, A.C., Judice, J.K.; Wells, J.A. (1994) Methods in Enzymology, 289, 300.
4. de Beer R. J, Nuijens T., Wiermans L., Quaedflieg P. J. L. M., Rutjes F. P. (2012) Org.
Biomol. Chem., 10, 6767-6775.
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Contact us
Customer Service/Ordering
Contact us
Technical support
Tel: +31 (0)46 47 60 675
Web: www.enzypep.com
e-mail: [email protected]
Notice to purchaser
OMNI Kit components may be used for research purposes only. They may not be used for any
other purpose, including, but not limited to, use in drugs, in vitro diagnostics purposes,
therapeutics or in humans. EnzyPep products may not be transferred to third parties, sold, resold,
modified for resale, or used to manufacture commercial products or to provide a service to third
parties without written approval of EnzyPep BV.
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