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Enzyme Labeled Peptide Synthesis

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Enzymes are most commonly used to detect proteins via direct and indirect antibody detection strategies or as nucleic acid hybridization probes and detection methods required for sensitive assays. CD Formulation offers custom enzyme-labeled peptide synthesis and modification services for use as signal-generating molecules. By using different conjugation chemistries, we can selectively conjugate enzymes to peptides without interfering with peptide activity and enable enzyme labels to be conjugated to specific proteins and peptides as a means of signal generation.

Introduction to Enzyme Labeled Peptides

Enzymes are widely used in molecular biology because they have specific properties that allow for the visual and spectrophotometric detection of these immune complexes. There is a growing awareness that enzyme conjugates created by conjugating enzymes to peptides using peptides as building blocks can serve as sensitive probes. Such enzyme labels offer the best combination of properties, with high sensitivity, low background, and rapid detection.

Fig. 1 The enzyme was immobilized on the peptide-modified surface through a specific reaction between the C-terminal cysteine and the maleimide-activated surface. (Fu J, et al., 2011)

Explore Our Custom Enzyme Labeled Peptide Synthesis Services

CD Formulation offers several custom synthesis options for peptides and enzymes listed below, but we are happy to discuss conjugation projects using your own enzymes or peptides.

Horseradish peroxidase (HRP)-peptide immunoconjugates.
Alkaline phosphatase (ALP)-peptide immunoconjugates.
Alkaline phosphatase amplification (AMP)-peptide immunoconjugates.
Penicillinase (PNC)-peptide immunoconjugates.
α-D-galactosidase-peptide immunoconjugates.
Glucose-t-phosphate dehydrogenase-peptide immunoconjugates.
Other enzyme-peptide immunoconjugates.
Non-enzyme protein immunoconjugates.

Choose the Right Preparation Method for Your Enzyme Labeled Peptide

Enzymes, those remarkable biocatalysts, can undergo intricate modifications to reactive moieties that conjugate with pre-activated peptide entities boasting an array of chemically reactive functionalities—think amines, thiols, carboxylic acids, hydroxyls, aldehydes, and ketones. Navigating through the labyrinth of conjugation chemistries, we're here to partner with you in the quest for the most fitting chemical method tailored to the unique specifications of your peptide masterpiece, encompassing a spectrum of innovative options to fulfill your project's requirements:

Crosslinker	Description
Glutaraldehyde	Glutaraldehyde is a homobifunctional cross-linker with aldehyde functional groups at both ends of the 5-carbon chain, which can react with amines to form secondary amine bonds.
Periodic Acid Oxidation	Periodic acid can oxidize enzymes such as GO and HRP to generate active aldehyde residues that can bind.
Hydrazide	This group can react with a carbonyl group to form a stable hydrazone bond.
SMCC (Succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate)	SMCC is a heterobifunctional crosslinker with N-hydroxysuccinimide (NHS) ester and maleimide groups that allows covalent coupling of amine- and sulfhydryl-containing molecules to form amide bonds for enzyme activation.
SPDP (succinimidyl 3-(2-pyridyldithio)propionate )	SDPP is a heterobifunctional crosslinker. It is commonly used in immunotoxins, antibody enzymes, and enzyme-labeled DNA probes.

How Do We Ensure Your Enzyme Labeled Peptide is of High Purity?

After labeling the enzyme with a cross-linking reagent, the final conjugate must first be separated from excess or unreacted reagents by gel filtration or dialysis. This typically involves:

Purification: Generally, our scientists use dialysis to remove unreacted reagents from the reaction solution. Techniques such as stirred cell filtration, tangential flow filtration (TFF), and gel filtration chromatography are used to remove excess reagents or separate and identify cross-linked products.
Isolation and Characterization: After the product is purified, our scientists perform different types of studies, including spectroscopy (MALDI-TOF, ESI, LC-MS fluorescence), electrophoresis, immunochemical, and enzyme analysis, for further characterization.
Quality Control (QC): We also provide an independent QC program to ensure that each conjugate delivered is of the highest quality.

Peptide Manufacturing & Analytical Services

In addition to peptide synthesis capabilities, CD Formulation combines flexible GMP manufacturing facilities with cutting-edge peptide analytical knowledge to provide a full range of quality control testing services to accelerate the commercialization of your products, including:

Peptide identification (ESI-MS).
Peptide Molecular weight determination.
Peptide sequencing.
Peptide quantification/peptide content determination.
Peptide purity and impurity analysis (HPLC/UV).
Amino acid sequence.
Amino acid composition determination.
Net peptide content.

Enantiomeric purity testing (GC/MS; LC).
Residual counterion testing (e.g. TFA).
Elemental analysis.
Residual solvent testing.
Water content testing (GC or KF).
Peptide solubility testing.
Peptide stability testing.

Optical rotation determination.
Bioburden testing(TAMC/TYMC).
Bacterial endotoxin testing.
Sterility testing.
Cytotoxicity testing.
Process/product related impurity testing.
Other pharmacopoeia testing.

Publication

Published Data

Technology: Enzyme-Catalyzed Technology

Journal: Chembiochem.

IF: 2.6

Published: 2009

Results:

The authors describe a synthesis of an alkyne-containing substrate consisting of nine non-hydrogen atoms. The substrate was synthesized in six steps from 3-methyl-2-buten-1-ol and enzymatically incorporated into proteins and peptides using protein farnesyltransferase. After prenylation, carboxypeptidase Y was used to remove the last three amino acids required for enzymatic recognition and leave one residue (cysteine in the "CAAX box") and the prenyl analog as the only modification. On this basis, the authors also studied and demonstrated the effect of this small tag on the target protein's solubility, which can minimize the modification's effect on solubility.

CD Formulation is a trusted partner for peptide synthesis. Please don't hesitate to contact us if you are considering using enzyme labeled peptides in your project. We look forward to cooperating with you.

References

Fu J, Reinhold J, Woodbury NW. Peptide-modified surfaces for enzyme immobilization. PLoS One. 2011 Apr 8;6(4):e18692.
Wollack JW, Silverman JM, Petzold CJ, et al. A minimalist substrate for enzymatic peptide and protein conjugation. Chembiochem. 2009 Dec 14;10(18):2934-43.

How It Works

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Submit a service request describing your specific research or development need.

STEP 2

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STEP 3

Execute the project with real-time communication, and deliver the final report promptly.

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