Peptide Synthesis

Severn Biotech's custom synthesis of peptides is available at extremely competitive prices. The quality of peptide is assured by the use of only the highest grade reagents. Quantities within the range 1mg-30g scale can be synthesised, and a wide range of modifications and conjugations are also available.

30g Peptide synthesis scale reactor

Purity Levels

The standard purity is > 85% which is sufficient for antigenic peptides for immunological applications. Ultra-pure peptides of > 95% are obtained by semi-preparative and preparative scale C18 reverse phase HPLC; recommended for bio-active and intracellular studies. All peptides are supplied with HPLC profile and Mass Spectromatogram for verification of purity and molecular weight. Net peptide content can also be supplied.

MALDI-TOF Mass Spec Verification

Additional Modifications to Peptides

• Acetylation - N-terminal
• Phosphorylation - available at serine, threonine and tyroseine residues
• Biotinylation - N-terminal or primary amine
• Cyclisation

Other modification are available upon request.

Conjugation to Carrier Proteins

Synthesised peptides can be conjugated to various carrier proteins (BSA, KLH, PPD, Thyroglobulin) using either hetero- or homobifunctional cross-linking reagents as appropriate. Peptides are coupled via a carboxy or amino terminal cysteine residue to available amines on the carrier protein. The product is lyophilised in a sodium phosphate buffer pH 7.1. When reconstituted in 2mls of water, the concentration is 50mM PO4 at pH 7.0.

Multiple Antigenic Peptides (MAPs)

Traditionally, small peptide fragments are attached to a large, inert carrier protein to generate an adequate immune response. The multiple antigenic antigenic peptide approach eliminates the need for a carrier protein. This is replaced by a branched chain poly-lysine core of low antigenicity. To this core can be added eight antigenic peptides. The result being a stable structure with a high ratio of peptide to core. The poly-lysine core is sufficient for attachment to plastic, thereby performing well in direct immunoassays.

Technical Section

PEPTIDES

Storage of Peptides

Custom-made peptides are supplied lyophilised to avoid degradation of the product. Most peptides, if stored tightly sealed and at -20°C in lyophilised form will remain stable for several years. To maintain stability, it is recommended that vials should be allowed to warm to room temperature in a dessicator with fresh dessicant before opening to prevent condensation forming on the product.

Peptides in solution are much less stable. Storage is recommended at -20°C in a buffered solution at pH 5-7. To avoid bacterial degradation, sterile purified water must be used for dissolution or the solution should be filtered using a 0.45m or 02m filter. The peptide stock solution should be aliquoted out before freezing to prevent damage caused by repeated freezing and thawing. After thawing, any remaining peptide in an aliquot should be discarded.

Dissolving Peptides

The secondary structure of peptides is often a hindrance to its dissolution, particularly with hydrophobic peptides. Since the formation of this secondary structure may be promoted by salts, the peptide should first be dissolved in sterile distilled or deionised water. At this stage the concentration of the peptide should be kept higher than the required final concentration to allow for addition of other solubilising agents if required and buffer salts which should be added only after the peptide is fully dissolved. Peptides containing cysteine, tryptophan or methionine are particularly susceptible to oxidation and should therefore be dissolved in oxygen-free water (prepared by degassing under reduced pressure or by sparging with an inert gas).

If the peptide does not dissolve in pure water, the following measures may be taken:

• Sonication. This may help to break up any particles and increase the rate of dissolution.
• Dilute aqueous acetic acid or ammonia(10%v/v) added dropwise to basic or acidic peptides respectively.
• If the above methods do not succeed in dissolving the peptide, addition of a chaotropic agent such DMF, Urea or Guanidium hydrochloride may be helpful. Addition should be dropwise until the peptide dissolves. These agents may interfere with biological systems so should only be used if the peptide is being prepared for analytical purposes.

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Custom Oligonucleotide Synthesis

Severn Biotech's oligonucleotides are synthesised by experienced and dedicated scientists with quality the highest priority. A wide range of labels, modifications and base substitutions are available, most of which are described here.

Each oligonucleotide undergoes UV analysis to assess the quantity produced and to ensure salt residues have been eliminated by the desalting process which is standard. Each oligonucleotide sequence is guaranteed to be correct and meet QC specification.

Delivery is within two working days from receipt of order with an extra day added on if any modifications or purification is required.

Scale of Synthesis

Synthesis can be performed at different scales normally 0.05M, 0.2M and 1.0M. Larger synthesis scales providing mg quantities necessary for in vivo work, are available upon request.

Estimated Minimum yield of Oligonucleotide (for oligos of average base length 20)
	Unpurified yield		Purified yield
	OD	g	OD	g
0.05M	8	160	2	80
0.2M	23	800	5	300
1.0M	60	4000	45	1500

1 OD unit is taken to be equivalent to 20g DNA

Purification

All oligonucleotides synthesised by Severn Biotech are desalted as standard. Further purification is available via either HPLC, C18 cartridge or PAGE, the level of purification varying between these different techniques as outlined below:

RNA Synthesis

RNA oligos are available at the 0.2M and 1.0M scales of synthesis. Standard length oligos (up to 20-mers) are generally synthesised, but longer oligonucleotides may be considered upon request. RNA molecules can either be supplied with or without a 2'-allyl protecting group. This protecting group greatly increases the stability of the RNA oligonucleotide during storage, but will need to be removed prior to use.

Technical Section

OLIGONUCLEOTIDES

Storage of Oligonucleotides

Oligonucleotides are delivered as a lyophilised pellet and should be kept in this form if long term storage is required. Shelf life if stored at -20° is over a year.

Working solutions of DNA should not be kept longer than overnight to avoid degradation of the product. Only sterile water and buffer solutions should be used. If storing as a solution, concentrations should be mMolar i.e. stock solutions and should be aliquoted out before freezing as repeated freeze/thaw conditions may cause degradation of the oligo.

If kept at -20°C storage is from three-six months. At 4° this is reduced to approximately two weeks.

The exact weight of an oligonucleotide (MWo) is as follows:

MWo = [No. of As x MWA] + [No. of Cs x MWC] + [No. of Gs x MWG] + [No.of Ts x MWT]

Approximate Conversion Factors

10D260 unit ~ 20g DNA

Approximate MWo = No. of bases 	x 310

Melting temperature (Tm) ~ [2°C x No. of As & Ts] + [4°C x No. of Gs & Cs]

Antiserum Production

Severn Biotech Limited provides a tailored contract antiserum production service. Peptides to be used may be synthesised by our own service or customer-supplied A protein screening service is available to determine the most antigenic fragments available for use as the antigen.

We offer standard antiserum production protocols, but will be happy to undertake other requirements as specified by the customer. ELISA analysis of pre-screened serum and titres is also offered.

Further details are available on application to our offices.

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