Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 411

With the deciphering of the human genome, theoretically it is possible to mutate
every gene and produce an equivalent protein product. With 20 naturally occur-
ring amino acids, the possible combinations are 20 n , where n equals the number of
amino acids in the polypeptide, overlooking alterations by amino acid deletion or
addition. Techniques like saturation mutagenesis have been used to meet these ends.
Deletion of one of the three glycosylation sites from TPA produced a molecule with
increased half-life and reduced clearance. Genetic engineering of serine proteases
yielded a series of proteases with amino acid structures attached to different func-
tional domains offering high structural specificity and selectivity.
Fusion of different gene segments has yielded proteins with novel combina-
tions of properties. For example, fusion of the truncated IgG heavy-chain gene to the
gene for a staphylococcal nuclease produced a protein with the retention of antigen-
binding specificity and nuclease activity. Chimeric or recombinant antibodies prepared
by this method can be used in the future for passive or active immunity. A plethora
of gene fusions and amino acid replacements have been reported for the production
of thermostable kanamycin nucleotidyltransferase. Alhough now we may be in a very
preliminary stage of protein studies, in the future it will be possible to predict three-
dimensional structures of proteins along with their physiological and biological activi-
ties. This is a tremendously fast-developing science that has led to leaps in the field of
medical science and therapeutics. In the future, it will be possible for immunologists
and allergists to understand the cellular and molecular basis of immunity. The mecha-
nisms of induced immunotolerance controlling the immune response to engineered
protein drugs are the next major challenge these scientists will have to tackle.
8.7 Protein Purification, Characterization, and Methods of
Analysis for Proteins
One of the major challenges for the peptide chemist is postsynthesis purification of
peptide derivative. Due to the inability of peptides of crystallization, purification
becomes a tough job. Racemization and formation of side products are two critical
areas that demand our major attention. Advances in the fields of science and technol-
ogy have rendered these tasks relatively simple and straightforward. In a solution-type
synthesis, peptide undergoes purification at multiple stages. However, purification
becomes a major issue when we talk about solid phase synthesis of peptides. Due to the
lack of purification of peptides in the solid phase synthetic procedure, the final product
ends up with shortened deleted peptides. One such example has been reported in solid
phase peptide synthesis of interleukin 3 (IL3), a 140 residue protein. Although, the final
crude product showed physical properties very much identical to those of the naturally
occurring protein, the quantitative assays showed that the synthetic material was from
0.5% to 30% as active as the original protein molecule. Purification was really trouble-
some and the use of highly sophisticated techniques could not resolve the issue. Hence,
the failure to resolve the problems of purification assumed serious proportions, with a
corresponding increase in the size of the synthetic protein. However, chromatographic