Molecular Biotechnology Core


CD Spectroscopy

A Circular Dichroisms (CD) Spectropolarimeter (Model J-815 from Jasco) is a type of light absorption spectroscopy that can provide information on the structures of optically active biological macromolecules. CD spectra of proteins between 250 and 185 nm can be analyzed for different secondary structural types such as, alpha helix, parallel and antiparallel beta sheet, turn and other random structures.

The CD spectroscopy is a shared resource and is available to researchers of Cleveland Clinic Foundation and neighboring Institutions. The details about CD spectroscopy are as follows:

CD Capabilities:

Model J-815 Spectropolarimeter (CD and ABS measurement modes) with computer controlled data acquisition; wavelength range 163-900 nm.
Single position Peltier system with Fluorescence capability.
Dual range titration system for automated pH, ligand and denaturant titrations.
Scanning Emission Monochromator allows spectral EM scans simultaneous with CD measurement (wavelength range 200-750 nm)
Temperature controlled circulator Julabo

CD Spectropolarimeter can be used for the following applications:

  • Secondary structure analysis of proteins in solution
  • Protein folding and conformational studies
  • DNA/RNA interaction studies
  • Temperature controlled kinetic studies
  • Thermal denaturation profiles of aqueous proteins

Isothermal Titration Calorimetry

What is ITC?

Isothermal Titration Calorimetry (ITC) is the gold standard for measuring biomolecular interactions. ITC simultaneously determines all binding parameters (n, K, δH and δS) in a single experiment information that cannot be obtained from any other method. When substances bind, heat is either generated or absorbed. ITC is a thermodynamic technique that directly measures the heat released or absorbed during a biomolecular binding event. Measurement of this heat allows accurate determination of binding constants (KB), reaction stoichiometry (n), enthalpy (δH) and entropy (δS), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment. Because ITC goes beyond binding affinities and can elucidate the mechanism of the molecular interaction, it has become the method of choice for characterizing biomolecular interactions.

Applications include:

  • Characterization of molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules.
  • Lead optimization.
  • Enzyme kinetics.
  • Assessment of the effect of molecular structure changes on binding mechanisms.
  • Assessment of biological activity.

Interactions between any two molecules can be studied with ITC, including:

  • Protein-small molecule
  • Protein-protein
  • Target-drug
  • Enzyme-inhibitor
  • Antibody-antigen
  • Protein-DNA
  • Protein-lipid
  • Small molecule-small molecule

Peptide Synthesis

Peptides are complex molecules and each peptide sequence is unique with regard to its chemical and physical properties. Peptides are synthesized by the solid-phase method using Fmoc chemistry using a liberty peptide synthesizer(CEM Inc.) based on microwave technology and an Omega 396 multiple peptide synthesizer (Advanced ChemTech).

Peptides are complex molecules and each peptide sequence is unique with regard to its chemical and physical properties. Peptides are synthesized by the solid-phase method using Fmoc chemistry on an Omega 396 multiple peptide synthesizer from Advanced ChemTech. The Core also uses an Applied Biosystems Model 431A Synthesizer for longer peptides. Synthesis is carried out at 50 µmole, 100 µmole or 250 µmole scales. On an average, a synthesis scale of 100 µmoles will yield about 50-70 mg of a 10-residue peptide and 100-150 mg of a 20-residue peptide depending on the substitution rating of the resin. The peptide synthesis service provides a complete spectrum of services that include, synthesis, cleavage, HPLC purification, peptide modifications, cyclization and mass analysis for quality assurance. Before submitting your peptide sequences to the Core, it is advisable to consider the following:

Synthesis is carried out at 50 µmole, 100 µmole or 250 µmole scales. On an average, a synthesis scale of 100 µmoles will yield about 50-70 mg crude peptide depending on the substitution rating and sequence of the of the peptide. The peptide synthesis service provides a complete spectrum of services that include, synthesis, cleavage, HPLC purification, peptide modifications, cyclization and mass analysis for quality assurance. The Core offers you an excellent service at competitive prices for the customized synthesis of modified and conjugated peptides. Before submitting your peptide sequences to the Core, it is advisable to consider the following:

Every peptide sequence synthesized is a unique one. Some peptides sequences are difficult to synthesize although, we attempt to synthesize every peptide sequence. Peptides with multiple cysteines, methionine, arginine and tryptophan residues can often be difficult to synthesize. Avoid a proline residue at the C-terminal of the peptide. Peptides will be deprotected and cleaved from the resin with 82.5-90% trifluoroacetic acid, partially purified, and lyophilized. The lyophilized material will be provided as a "crude" preparation. Turnaround time is about one week to three weeks depending on the length of the peptide, customization and purity level. Some of the modifications are as listed below:

Acetylation, acylation, biotinylation and amidation
Thioethers, hydroxyethylene isosteres, retro-inverso peptides
Cyclizations using disulfide bond (S-S bond)
Phosphorylation, sulfatation and nitrosylation at tyr
Incorporation of chromophores
Fluorescent dyes/fluorogenic groups, fluorophore/quencher pairs
Conjugation to a carrier protein
Incorporation of D-enantiomers and unusual amino acids
Labeling with stable isotopes (15N and 13C)
Introduction of C-terminal alcohol moieties, ester and thioester groups
Incorporation of Azido and Alkyl groups
Stabilizing modifications including PEGylation, N-methylated amino acids and reduced peptide bonds
Incorporation of Thr- & Ser-monosaccharide residues (glycosyl residues)

Quality Control Check

Strict quality control in peptide synthesis is the most important part. Besides synthesis related problems, there are post-synthesis adduct formation & modification problems associated with peptides. An analytical-scale reverse-phase HPLC chromatogram and the mass analysis by either MALDI-TOF-TOF mass spectrometry will be provided for every peptide to ensure the highest quality of synthetic product. The Core will also get the amino acid analysis performed from an outside facility if so requested.

Modification of Synthetic Peptides

The core laboratory routinely synthesizes peptides with the following modifications: peptides containing unusual, non-protein or modified amino acids (e.g., phospho-Tyr, phospho-Ser/Thr, D-amino acids or other analogs, Thr-glycosyl)), biotinylation at N- or C- terminals, fatty acid conjugation (butyric, hexanoic, myristic, palmitic), fluorochrome conjugation (fluorescein, rhodamine, phycoerythrin) N-acetylation, peptides amides, and peptide cyclization via s-s bonds.

Peptide Purification

A successful synthesis is dependent on the sequence of the peptide. Likewise the coupling efficiency of amino acids in the sequence and ultimate purity of the synthesized product is also related to the sequence. Impurities such as salts, deletion peptides (created due to incomplete coupling), protecting groups and modified peptides can range from 10-50% by weight. It is recommended that all the peptides should be purified prior to use. Purification of synthetic peptides is carried out on a semi-preparative Waters HPLC system using a RP C-18 column (2.2 cm x 25 cm). Fractions containing the peptide will be collected and lyophilized. In general, 50 mg to 70 mg of crude material will be injected at a time to obtain 20 mg to 50 mg of purified peptide at a minimum purity of 95%. The actual purity obviously depends on how conservatively the fractions are pooled. Users need to specify what level of purity is required.

Peptide Storage

Peptides are provided as lyophilized product that should be stored as dry powder at -20°C to -70°C in a desiccator, if possible. After lyophilization, peptides retain significant amounts of water. Peptides are slowly degraded particularly the cysteine-containing peptides are oxidized over time at -20°C. The water content of a given peptide powder varies depending on sequence of the peptide, storage conditions, handling, and other factors. Some peptides are very hygroscopic. A lyophilized peptide also contains counter-ions that are paired with charged residues (e.g., acetate or trifluoroacetate for positively charged residues). The presence of counter-ions cannot be avoided because of ion pairing. Water and counter-ion content in a given peptide product vary widely and combined together may be as high as 30%. The exact concentration of the peptide in the crude product can be determined by amino acid analysis using a small aliquot from the crude peptide solution. The volume of this aliquot must be accurately measured and recorded because this value is necessary for calculating the concentration of the stock-solution. Peptide solution is much less stable than the lyophilized products. The peptide solution can be stored with the following guidelines:

  • The peptide solution should be stored at -20°C in small aliquots. The stock solution should be aliquoted upon arrival to prevent degradation caused by repeated freezing and thawing.
  • Pass the peptide solution through a sterile filter before storing to prevent bacterial contamination
  • Maintain peptide in an oxygen-free environment as peptides having cysteine, methionine, trypophan, glutamine, and asparagine residues are susceptible to oxidation and have reduced self-life.
Reconstitution of Lyophilized and Frozen Peptides

When using frozen peptides, the vial or tube should be allowed to warm to room temperature in a desiccator containing fresh desiccant before opening. Failure to do this can cause condensation to form on the peptide when the vial is opened and will affect the stability of the product. The best solvent to reconstitute the peptide is DMF (up to 30%), added drop wise until the peptide dissolves.

Peptide Conjugation to a Carrier Protein for Immunization

The sequence of an antigen peptide should be designed such that the peptide can be attached to a carrier-protein (e.g., keyhole limpet hemocyanin, ovalbumin, bovine gamma-globulin, or bovine serum albumin). Peptides can be conjugated to these carrier reagents through either its amino- or carboxyl-terminal ends. This type of cross-linking to C- or N- terminal can be performed via a pre-activated carrier protein. The commonly used cross-linking reagents are: MBS (m-maleimidobenzoyl-N-hydrosuccinamide) cross-linker to C, K or free amino groups; EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride cross-linker to Asp, glu or a carboxy terminus. The extent of coupling will be estimated by amino acid analysis.

The Multiple Antigen Peptide System

The Multiple Antigen Peptide (MAP) concept was introduced in 90's to avoid chemically undefined entity of an antigen-carrier system. The MAP system utilizes a peptidyl core of three or seven radially branched lysine residues, on to which the antigen sequences of interest can be built using standard solid-phase chemistry. The lysine core yields the MAP bearing four or eight copies of the peptide epitope (high molar ratio), depending on the inner core that generally accounts for less than 10% of total molecular weight. The MAP system does not require a carrier protein for conjugation. The high molar ratio and dense packing of multiple copies of the antigenic epitope in a MAP has been shown to produce strong immunogenic response. Synthesis of a multiple antigenic peptide is carried out at a 0.1 mmol scale. This ensures maximum coupling yield during synthesis and, hence, optimizes the preparation.

Storm 820 Phosphorimager

The Storm system is an optical scanner that produces digital images of radioactive or fluorescently labeled samples. The Storm 820 Phosphorimager is used only to scan a phosphor screen that has been exposed with a radioactive sample. Users will need to provide their own phosphor screen. One can analyze the results of a scan with ImageQuant TL which has been installed on the data acquisition computer. Storm 820 is located in NB-5, near the elevators. A sign-up log book is available next to the instrument.

Surface Plasmon Resonance (SPR)

Surface Plasmon Resonance (SPR) has been used to monitor macromolecular interactions in real time. The Biacore 3000 system is an instrument that uses SPR technology for measuring the interactions of macromolecules with each other, and with small ligands. One of the ligands is immobilized on carboxymethylated dextran over a gold surface, while the second partner (analyte) is captured as it flows over the immobilized ligand surface. Most ligands can be directly immobilized onto the surface of the chip via amino groups, carbohydrate moieties, or sulfhydryl groups. Others are immobilized indirectly through the use of biotinylation of the ligand (such as biotinylated peptides or oligonucleotides), or through immobilized monoclonal antibodies (such as anti-GST). Typical amounts of a protein ligand needed for an immobilization reaction is about 1 µg. The immobilized ligands are remarkably resilient and maintain their biological activity.

Biacore 300

Biacore can be used for measuring the binding constants of macromolecular interactions (protein-protein, nucleic acids - protein, protein-ligand etc) and in many other applications such as, epitope mapping, ranking antibody affinities, concentration measurements, characterization of mutant proteins and Ligand fishing.

The bound analytes can be stripped from the immobilized ligand without affecting its activity to allow many cycles of binding and regeneration on the same immobilized surface. Interaction is detected via SPR, in real time, at high sensitivity, without the use of radioactivity. The equilibrium rate constants KA (strength of binding and KD (affinity) are measurable in the range of 105 - 5x1012 [M-1] and 10-5 - 5x10-12 [M], respectively. Because the same affinity may reflect different on-rates and off-rates, this instrument excels over most other methods of affinity measurements in that it measures on-rates (ka in the range of 103 - 5x107 M-1s-1) and off-rates (kd of 10-6 - 10-2 s-1). Concentration determination experiments are also feasible.

The instrument and services are available on a fee-for-service basis to CCF researchers as well as investigators from CWRU and other area institutions. The sign-up sheet for scheduling your time on the instrument is available from the Molecular Biotechnology Core Laboratory.