Custom Peptide Synthesis

We had produced the peptides containing 200 residues. However, peptides longer than 100a.a are extremely difficult to synthesize. We can evalute them case by case and try our best to make them by fragmentation and ligation.

We had produced peptides containing 16 consecutive Pro residues. If you'd like to, we can have a try, but it is hard to ensure success.

For the sequence with "Q" at the N terminus, it is important to note that this configuration is not stable and may undergo a cyclization reaction to form pGlu. While obtaining a qualified peptide with this sequence can be challenging, we are willing to make an attempt if you are interested. However, if the peptide is urgently needed for your project, you may consider the following options to prevent such cyclization:
Removing "Q" from the N-terminus
Adding another amino acid in front of "Q"
Applying N-terminus acetylation.

Unless otherwise specified, custom peptides are provided as lyophilized powder in TFA salt (trifluoroacetate) form. Due to variations in synthesis or purification processes, some peptides may contain other counterions.

To ensure experimental consistency and minimize TFA interference, we offer two TFA exchange services:

• Standard TFA Exchange Service: reduce TFA level to be less than 10%
• Guaranteed TFA Exchange Service: guarantee TFA level to be less than 1%

For special requests or further questions, please contact: [email protected].

In chemical terms, a counterion is an ion of opposite charge to another ion in a solution or an electrochemical system. In peptide synthesis, counterions such as TFA are used for cleavege and HPLC separation, and TFA counterions binds to the basic amino acids or N-termini, so synthetic peptides are usually obtained as TFA salts. In some cases, TFA counterions may be harmful to biological assays (see Top 5 reasons peptide assays fail), however GenScript can replace TFA counterions with acetate or HCl, upon requesting our TFA removal service.

It is important to understand the difference between net peptide content and total peptide content. The dry peptide powder shipped to you usually contains not only peptides, but also some other substances such as water, absorbed solvents, counterions, and salts. The total peptide content(gross weight) refers to the weight of this mixture. Net peptide weight indicates the actual weight of the peptide component of your sample. Net peptide content is usually 50-80% of the total peptide weight (also called gross peptide weight) and is usually determined by amino acid analysis or Nitrogen analysis. Net peptide content should not be confused with purity. Purity defines the percentage of the target peptide sequence in the peptide component of your sample.

Theoretical net peptide content (calculated assuming that counterions are the only non-peptide components present in your peptide sample) can be estimated by dividing molecular weight of the peptide by a sum of this molecular weight and a number of trifluoroacetate counterions that are required to neutralize the peptide multiplied by the molecular weight of the TFA counterion (MW= 114). For example, a synthetic peptide of MW=1000 with a free N-terminal amino group and one Arg has theoretical net peptide content of 1000/(1000 + 2 x 114 ) = 1000/1228 =0.81 or 81%. In practice, counterions are not the only possible non-peptide impuritiies in the peptide sample. It can also contain water, absorbed solvents and traces of other substances. As a result, the actual net peptide content is usually determined by quantitative amino acid analysis.

Highly hydrophobic peptides are prone to aggregate, which increases the difficulty of synthesis. Highly hydrophobic peptides are easy to adhere to the column, which increases the difficulty of purification.

For the purity that is less than 70%, GenScript would set it as crude.

An appropriate peptide purity is a critical factor for the success of a scientific experiment. In general, the higher the peptide purity level, the more favorable the experimental outcome. Impurities in lower purity peptides may result in toxicity or unwanted side reactions, especially for some applications such as animal studies where exceedingly pure peptides (>98% purity) are required. More details can be found at this link: https://www.genscript.com/peptide-purity.html

In general, GenScript will lyophilize the target fraction in purification directly, the main reagents are the ultrapure water, acetonitrile and TFA in mobile phases.

Peptides are delivered in lyophilized powder form by default, aliquoted in individual sample tubes. Meanwhile, peptides can be delivered according to customer’s requirements.

A regular peptide label contains peptide name, peptide service type, quantity, purity and Lot number. If extra information is needed, such as sequence or molecular weight, please kindly add this request when making the quote, GenScript can provide a customized label.

In general, the appearance of a peptide is closely related to the peptide sequence itself, it is not recommended to do comparison between different peptides. Besides, peptides delivered as white lyophilized (freeze-dried) powder can differ in visual appearance between peptides due to the retention of different compositions in the final sample. For example, the freezing cycle can contribute to visual differences depending on the rate; a slower rate will compact the peptide into a dense form. During the drying process, more water may evaporate, leaving very little peptide visible to the naked eye. The pressure applied in the process will differ from lot to lot as well. These possible scenarios may change the appearance of peptide sample in the vial but do not change the amount of peptide contained in the vial, which is weighed via analytical balance.

The delivered peptides are accurately weighed and aliquoted, if the peptides are invisible, there are several reasons for your reference:

1. The peptide amount is too small to be visible using naked eye. It is recommended to dissolve the peptide in the vials provided by GenScript, then perform the experiments directly.

2. Peptides might be adhered to the vial wall or cap, it is recommended to perform centrifugal or shake operation before your experiments.

3. Hydrophilic sequences absorbed water during the transit or storage, it is recommended to re-lyophilize the peptides before experiments.

There are three common shipment temperatures: room temperature, blue ice (approximately 4 degrees) and dry ice (approximately -80 degrees).

Without specific requests, peptides in lyophilized powder format will be sent out at room temperature, and peptides in liquid format will be sent out with dry ice.

If there are special requests, please kindly add the note when preparing the quote or let GenScript know.

Adding PEG or several Lys (3~4) in the sequence may improve the hydrophilicity of the peptides.

Our typical turnaround time is about 2-3 weeks. The turnaround time may vary depending on the peptide length and complexity of synthesis.

GenScript usually use SPPS chemical methods for peptide production, but other strategies, such as LPPS, fragment condensation, native ligation or our proprietary recombinant method may also be used for peptide production.

Each peptide has its specific characteristics and not all outcomes can be anticipated. If something goes wrong during the synthesis and we cannot deliver your peptide on time, our project manager will contact you to tell you status and discuss the project with you.

Peptides are synthesized from the C-terminus to the N-terminus of the sequence.

SPPS is the most commonly used peptide synthesis method today, the C-terminus of the first amino acid is coupled to an activated solid support (resin). Peptide synthesis occurs by coupling the carboxyl group of the incoming amino acid to the N-terminus of the growing peptide chain. Fmoc (9-fluorene methoxycarbonyl) is used to protect α-amino groups, it is stable under acid conditions and can be quickly removed under alkaline conditions.

Boc is also N α-position protecting group for SPPS. Boc amino acid is attached to an insoluble support resin via a HF cleavable linker. Deprotection of Boc, is accomplished by treatment of the amino acid with TFA. The second Boc amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the solid support via HF cleavage. However, both N α-position protecting group and side chain protecting groups are acidic removable, and rounds of strong acid treatment would degrade the peptide, so this method is not very commonly used.

The peptide conjugated the KLH that is based on a mass ratio of 1:1, the peptide is quite excessive in this reaction, so the peptide is indeed coupled on KLH.

Yes, the resin peptide GenScript provided is synthesized via solid phase peptide synthesis and was not cleaved from the resin. The resin type includes 2-Chlorotrityl chloride (CTC) resin, Rink-NH2 resin and Wang resin. And the protecting groups of amino acid side chains are not removed. In general, N-terminal Fmoc would be removed, if not necessary, please let us know when preparing the quote. But as the peptides are on the resin, they cannot be purified.

Peptide synthesis protocol: The general protocol of Fmoc synthesis: The first Fmoc amino acid is attached to an insoluble support resin via an acid labile linker. Deprotection of Fmoc, is accomplished by treatment of the amino acid with a base, usually piperidine. The second Fmoc amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the solid support via TFA cleavage. Disulfide bond formation: Usually a peptide containing disulfide bonds is first assembled in a linear form with Cys in its sequence. The disulfide bond is formed after synthesis via solution phase, oxidative reagents, such as oxygen (air).

There is a certain spatial folding of the peptide, which causes that next a few amino acids cannot be effectively coupled.

GenScript will try to perform double coupling, microwave or/and increase the material amount, prolong the reaction time, and reduce the substitution degree of resin to alleviate this situation.

Trifluoroacetic acid (TFA) is a strong acid, which is commonly used to cleave synthesized peptides from solid-phase resins and is also used to improve HPLC performance in the peptide purification step. Although TFA is important, residual TFA in custom peptides can cause inexplicable discrepancies in subsequent assay data. If needed, please kindly download GenScript’s Review to get more idea about Impact of TFA in below link: https://www.genscript.com/review-impact-of-tfa-in-peptide.html

In general, most custom peptides are delivered as lyophilized TFA salts, and can contain as much as 10%-45% TFA. To improve the study outcome, TFA removal is essential before starting your experiment.

GenScript purifies and analyzes its synthetic peptides via reverse-phase HPLC.The impurities are most likely salts and water, solvents, TFA, deletion, truncation, incompletely deprotected sequences, etc.