Peptide Synthesis: design and structure
In this article we detail some of the design considerations we use in peptides synthesis.
Design and structure of peptides Synthesis Options and Applications
We can incorporate all the standard amino acids and • Peptide antigens for antibody generation. Synthesis of peptide to target epitope, incorporation of post- other moieties from the extensive range of building translational modifications into a sequence. blocks available. We only use high quality reagents •Affinity columns for antibody purification. sourced from reputable suppliers to ensure our Fast, robust, effective peptide product for the purification of customers receive products of high quality. Synthetic peptide and protein antibodies from serum. difficulty is sequence dependent, however, our • Linear peptides. Syntheses from tri-peptides up to 90+ amino acids. peptide chemists, using their experience in synthesis • Branched peptides. Branching from a C-terminal optimisation, have increased our success rate of poly-lysine core or mid-sequence branching. This would synthesising difficult peptides e.g. amyloid peptides enable you to have individual peptides on different branches. and proteins. • Modified amino acid peptides. Incorporation of D-amino acids, phosphorylated amino acids, amino acid analogues, spacers. • Tags. Biotinylated, fatty acids, pegylation, farnysyl, Length of peptides photolabile linkers, maleimide. • Fluorescent and Dye Labelled Peptides. Our synthetic methods are routinely used to produce Peptides with fluorescent or other dye labels provide useful peptides and proteins in the range of 3-70 amino tools for monitoring biological interactions, receptor-ligand binding, protein structures and enzyme activity. acids. However, we also have experience of • Isotopically labelled peptides. successfully making peptides of over 80 amino acids. For use in quantitative mass spectrometry. Peptides are The longer the peptide the greater the number of synthesised incorporating amino acids enriched with the impurities to be removed from the target sequence, stable isotopes 13C and 15N. thereby affecting the absolute purity of the product. • Cyclic peptides. Cyclisation of a peptide provides the benefit of constructing a A longer peptide will also have a higher chance of constrained shaped peptide. containing a sequence region that is difficult to • Stapled peptides. synthesise, however with many years of experience Provide stable helical structures, aiding protease resistance AltaBioscience has overcome such challenging and cell-permeability and increasing binding affinity to target. peptides. We can advise on any possible concerns • Histone peptide synthesis and microarrays. • Custom microarray peptides. regarding the viability of a particular synthesis. For investigation of protein-peptide interactions using your own peptide sequences. • Peptide conjugation. o Peptide - protein conjugation KLH, BSA, DNA-hybrids. o Peptide - DNA conjugation DNA-hybrids via thiol or amino linkage.
Peptide synthesis in our Worcestershire laboratories
Design considerations Some of the following considerations may be helpful when determining peptide design:
A peptide with no charged or polar groups may be very insoluble. These amino acids decrease solubility: - Trp, Val, Ile, Phe. These amino acids increase solubility: - Lys, His, Arg, Asp, Glu, Ser, Thr. Proline breaks up beta sheet formations and although non-polar, helps to solubilise peptides. A spacer between a dye or tag and the rest of the peptide sequence can be advantageous. It is always more cost effective to put a dye or tag at the N-terminus rather than the C-terminus as this can be incorporated during the standard synthesis. C-terminus additions require many more steps, thus increasing the cost. N-terminal glutamine (Gln) should be avoided. It is very unstable and rapidly forms the cyclic pyro glutamic acid. It is best to add either pyroglutamic acid itself or include an acetyl group at the N- terminal glutamine. Avoid regions containing long strings of valine or isoleucine as these are extremely difficult to incorporate and may prevent any extension of the synthesis. Multiple additions of phospho-amino acids can sometimes be difficult to incorporate during synthesis. Naturally cysteine occurs in proteins with a disulphide bridge, therefore we would advise to avoid cysteine when designing peptides for raising antibodies.
Amino acid classification
The following table gives a general classification of the amino acids
Class Amino Acid Acidic, polar Asp, Glu Asn, Basic, polar His, Lys, Arg Polar uncharged Asn, Cys, Gly, Gln, Pro, Ser, Thr, Tyr Nonpolar and hydrophobic Ala, Ile, Leu, Met, Phe, Trp, Val
Methodology
We use solid phase synthesis to synthesise peptides (SPPS0, pioneered by Robert Bruce Merrifield). The process involves anchoring the C-terminal of the first amino acid to polystyrene based resins and then, in a stepwise process, coupling the carboxyl group or C-terminus of the next amino acid to the amino group or N- terminus of another (peptide bond formation). Due to the possibility of unintended side chain reactions, protecting groups are used where applicable.
Our use of Fmoc chemistry enables greater flexibility in synthesis options. Chemical peptide synthesis starts at the C-terminal end of the peptide and ends at the N-terminus.
When the peptide chain is complete, it is cleaved from the resin with acid, a process that also removes the amino acid side chain protection. After removal of the acid, the peptide is ready for QC by HPLC and mass spectrometry before being freeze dried, packaged and dispatched.
We are always keen to discuss a particular requirement for peptide synthesis. Future articles in this series will discuss peptide purity and analysis of the synthesised product; modifications and unnatural amino acids; specific options in making peptides for raising antibodies as well as considerations on solubility and storage.
For further information please contact us:
T: +44 (0)1527 584495
Author: Sat Sandhu, Principal Peptide Chemist.