What Amino Acids Will Cause Problems in the Synthesis of My Peptide?
One way to control the quality of the starting material (i.e., crude peptide) and thus improve the yield and ability to purify the peptide is to carefully choose the overall amino acid composition. This is an important peptide design issue that is frequently overlooked. Although the primary sequence of the peptide may be based upon the sequence of part of a native protein, there is still some leeway in the final peptide design. Since peptide solubility is strongly influenced by the amino acid composition, one tries to avoid designing peptides with a high content of hydrophobic residues, such as Leu, Val, Ile, Met, Phe and Trp. These peptides will be difficult to solubilize in aqueous solution or in some cases might be completely insoluble. This creates several problems, including difficulty purifying the peptide as well as issues with its solubilization in a suitable (i.e., aqueous) medium. Thus we advise customers that peptides should be designed with a hydrophobic amino acid content below 50%, and in addition, that at least 20% of the amino acid residues should be a charged amino acid. At pH 7.4, Asp, Glu, Lys, and Arg all have charged side chains. In some applications, charged amino acids can be added to either side of the peptide, or the ends of the peptide can be left unmodified (i.e., as a primary amino and/or carboxylic acid).

Other problems include trying to manufacture peptides containing multiple Cys, Met, or Trp residues. Because these residues are prone to oxidation, they present a problem for purification. The options would include trying to substitute other amino acids for Cys, Met or Trp, or alternatively, using analogues that are blocked to avoid the oxidation. Common substitutions include Norleucine for Met and Ser for Cys.

Beta-sheet formation is another important structural issue that one tries to avoid when designing peptides. When these structures arise, the peptides that result can have deletions and even truncations, resulting in a poor synthetic yield of the desired product. Peptides or regions of peptides with neighboring Val, Ile, Tyr, Phe, Trp, Leu, Gln, and Thr should be avoided, as these are the amino acids responsible for beta-sheet formation. There are some substitutions that can be made if these regions cannot be avoided.

In cases where the specific primary sequence of a peptide is required (e.g., producing an antipeptide antibody), the immunological potential of the region chosen (see Antibody FAQs) as well as its homology to other known proteins will often be the determining factor in choosing a sequence to be used. Given that most analyses used to find suitable peptide sequences will in fact choose peptides based on the presence of charged residues as well as prolines and other motifs, the presence of hydrophobic regions are rarely a problem for such applications. The presence of more than one cysteine, should be avoided. In cases where the sequence does contain two or more cysteines, a conservative substitution might be made, the peptide region might shifted up or downstream, or one or more cysteines might be blocked to avoid disulfide-bond formation.



 

How Do I Solubilize My Peptides?
What Amino Acids Will Cause Problems in the Synthesis of My Peptide?
What Purity is Right for My Peptide?
How Do I Choose a Peptide Sequence?
How Difficult is it to Synthesize a Custom Peptide?

 

 

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