CSEM Scientific and Technical Report 2008

A Universal Protein Assembler
H. Zepik
Chemical protein synthesis: a new assembler molecule for the ligation of appropriately functionalized peptides in solution. The same novel
approach can also be used for the purification of proteins, which is a very relevant problem today in the pharmaceutical industry
Many powerful drugs are protein-based. Examples include
hormones like insulin, interferons, monoclonal antibodies,
blood clotting factors. Proteins are chains of amino acids, from
a few tens to several hundreds. In recent years active
pharmaceutical ingredients (APIs) based on proteins have
received increased attention from the pharmaceutical industry.
Peptide and protein therapeutics are attractive due to their
high specificity and potency and low incidence of toxicology.
In contrast to small molecule drugs, protein drugs are rarely
produced by chemical synthesis. The major production
methods are extraction from natural sources and recombinant
DNA technology. However, chemical protein synthesis offers
several advantages. It is possible to modify the chain of amino
acids basically at-will, e.g. to insert unnatural amino acids with
specific properties, to introduce various labels, to attach
polymer- and sugar-chains. In addition the final product will
not be contaminated by any undesired biological material.
The chemical synthesis of peptide, has made enormous
progress in the last decades and it is now possible to make
peptides of up to ~50 amino acids reliably and in good yield.
The accumulation of side products and aggregation prevent
the synthesis of longer peptides and proteins. However, most
protein drugs, like monoclonal antibodies, have more than
100 amino acids.
To overcome this size limit, peptide segments can be ligated
in solution (Figure 1). However, due to the many reactive side
chains present in proteins, exclusive formation of the desired
bond only is impossible to achieve, unless a chemoselective
condensation is employed. This last approach has been
pioneered successfully but is restricted to certain amino acids
due to the chemistry.
NH 3 +
NH 3 +
SH
SH
NH 3 +
NH 3 +
OH
OH
CO 2 -
CO 2 -
CO 2 -
O
C
Figure 1: Unprotected peptide segments in solution cannot be ligated
efficiently due to many reactive side chains.
In order to overcome this strong limitation an alternative
approach which should ligate any peptide segment is being
developed. It is based on a dimeric “host”-molecule, the
assembler ‘A’, which recognizes and binds specific chemical
“guest”-groups. These groups are attached to both ends of the
ligation site (Figure 2). The role of the assembler molecule is
to bring the two ends into close proximity to increase their
specificity and rate of reaction. The “guest”-groups can be
NH
NH 3 +
OH
OH
SH
SH
NH 3 +
NH 3 +
CO 2 -
CO 2 -
CO 2 -
CO 2 -
attached to many different sites, which makes this assembler
universally applicable to all possible ligation sites.
NH 3 +
NH 3 +
Figure 2: Two functionalized peptide segments are bound by the
assembler ‘A’ whereby the carboxy- and amino-termini are brought
into close proximity resulting in a fast and specific ligation.
The “guest”-groups can then be removed to recover the final
protein product. While the attachment and later removal of the
functional groups is an additional manipulation and potential
complication, it offers a unique opportunity for the purification
of the desired protein. Immobilizing the assembler “host”molecule
on a solid support allows the selective binding of the
“guest”-decorated product protein (Figure 3). Since it carries
two binding sites it will bind much stronger to the support than
the starting peptides or any other additives present. The latter
can then be washed away, and the final protein will be
obtained, after removing the “guest”-groups, in pure form.
NH 3 +
SH
SH
SH
NH 3 +
NH 3 +
NH 3 +
A
OH
OH
OH
CO 2 -
CO 2 -
CO 2 -
CO 2 -
O
O
C NH
solid support
Figure 3: Strong binding of the product to the assembler immobilized
on the solid support.
As a conclusion, this novel approach to the ligation of peptide
segments can not only be used for the synthesis of arbitrary
protein molecules but it is also a promising alternate technique
for protein purification, for example for APIs.
NH 3 +
C NH
OH
OH
OH
SH
SH
SH
NH 3 +
NH 3 +
NH 3 +
CO 2 -
CO 2 -
CO 2 -
CO 2 -
CO 2 -
CO 2 -
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