|
|
PEPTIDE SYNTHESIS
The principal objectives of
peptide synthesis are to provide CURE investigators with (1)
synthetic peptides, (2) design strategies for distinctive peptide
reagents, and (3) state-of-the-art purification and analysis
of synthetic peptides. Specialized services include synthesis
of unusually long peptides (longer than 30 residues), synthesis
of peptides with unique amino acid analogs, and the availability
of a professional synthetic peptide chemist to help design peptides
that are best suited for specific research needs. The director
of the Core assists investigators in designing peptides that
meet the requirements of their particular study and advising
them on whether their peptide utilization is more suited for
large or small-scale synthesis. The Core has just obtained a
state-of-the-art peptide synthesizer that monitors deprotection
steps (ABI 433A) which replaces the older ABI 430 synthesizer.
This new instrument is especially suitable for large amounts
of peptide or for long peptides that are difficult to synthesize.
The Core has operated a multiple peptide synthesizer (ACT 496)
for the past five years and has become efficient at synthesis,
purification, and characterization of 10 to 20 peptides/week
through automation of necessary equipment.
After synthesis the peptides
are removed from the resin and unblocked with 90% trifluoroacetic
acid containing appropriate scavengers. The crude peptides are
precipitated with methanol, washed, and dried. The dry product
is dissolved and purified by reverse phase HPLC. A step that
is unique to the Core is that each fraction is then evaluated
by HPCE. This allows the detection of impurities that may not
be observed by analytical reverse phase HPLC. The importance
of this step has been demonstrated repeatedly in our laboratory.
Peptides that have greater than 95% purity when analyzed by reverse
phase have been shown to be less than 25% pure when analyzed
by HPCE. Peptides accompanied by certificates of purity from
several commercial or university peptide synthesis facilities
have been shown to contain major impurities by HPCE that were
undetected by HPLC. Our Core's analysis by HPCE adds a dimension
of quality control not found in most other sources of synthetic
peptides.
For collaborative studies, investigators
pay for synthesis, purification, and characterization of reagents.
|
|
