Enhanced Diagnostic Tools
The proper selection of a given peptide can enhance its yield and purity. One has to take into account the sequence itself, the length of the peptide and particular sequence repeats that will influence whether correct assembly and purification of said peptides are feasible. These parameters will determine the solubility of the peptide product. The following paragraphs, emphasize some key points in the proper design of a peptide prior to synthesis. Our peptide chemists will be more than happy to assist you in evaluating and recommending then best strategies for your peptide synthesis projects or visit our Free Gizmo Peptide Online Designing Software.
The majority of biological interesting peptides are derived from N-terminal, C-terminal, or internal sequences of native proteins. Structurally and geometrically it is known that the correct spacing of the amino acids is more important than the actual nature of the residues making up the linear sequence (except charged residues which are usually contact points of interaction). Therefore we have so called essential and non-essential residues; depending upon each sequence one can choose to replace small residues such us Gly for Val for example. Most often substitution or additions of amino acids are done for particular purposes, for example: Cys is added at either amino or carboxyl end for purposes of conjugation; a basic residue can be substituted for an acidic residue as a way to disrupt function ( and to prove the essentiality of said residue) ; a heavy isotope can be introduced( N15 instead of N14) as a way to follow by NMR interacting residues. The peptide ends can be capped by acetylation or amidation to mimic more closely the native sequence (and for stability) You get the idea that there are many reasons for modifying a given peptide sequence in order to better accommodate your particular objective. More recently methylated Arg and Lys are being used in the field of epigenetics, where it is thought that histone composition, play a role in protein expression. Please feel free to consult us for your own particular peptide applications.
Shorten the length of the sequence
As peptide length increases, the yield and purity of the crude product becomes lower. Most peptides of 15 residues or fewer can be synthesized without major difficulty. In addition, peptides of 10-15 residues in length are satisfactory for raising antisera to linear epitopes of intact proteins.
The better the solubility the easier it is to purify peptides. Peptides with a high content of hydrophobic residues( >50%), such as , Val, Leu,Ile Ile, Met, Phe and Trp, will either be either insoluble or have poor solubility in aqueous solution . This affects their utility in biological assays as it will be difficult to solubilize them. Ideally one would like to have at least one charged amino acid for every five residues . At pH 7 Asp, Asn, Glu,Gln Lys, and Arg all have charged side chains. Adding polar tails at either or both ends can improve solubility. Depending upon the application we can also use solubility enhancers ( linkers containing repeated ethylene glycol units).
Easily oxidazable residues such us Cys, Met, or Trp present in multiple positions residues are difficult to obtain in high purity. One should minimize, whenever possible the presence of these residues; a sequence containing multiple adajacent Trp residues will be a challenge and unless no other peptide choice is available , one may be better off avoiding said sequence. Also, conservative substitutions can be made for some residues; for example, Norleucine can be used as a replacement for Met, and Ser can be used as a less reactive replacement for Cys. The presence of certain repeats such us Val Gly Asp ( among others) along a sequence should be avoided, as these repeats tend to cyclize, reducing significantly the overall yield and purity of the desired full length peptide.
Peptide aggregation (ß sheet formation) is a problem encountered when multiple or adjacent residues such us Val, Ile, Tyr, Phe, Trp, Leu, Gln, and Thr are present along the sequence; ß-sheet formation causes aggregation, which results in poor solvent /reactant accessibility on the growing NH2 terminus . This problem can be circumvented by avoiding repeat/adjacent sequences above mentioned; also one can disrupt such stretches by inserting Pro( which acts as a swivel, allowing greater freedom of rotation) and Gly at every third or fourth residue of the peptide sequence.
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