Liutkus, Mantas
Description
A new method for the production of peptides through biological
expression was developed, utilising lactonisation-prone
chlorinated amino acids for latent peptide bond digestion.
Incorporation of halogenated amino acids into proteins is
possible due to the inherent inability of the biological
synthetic machinery to discriminate against compounds
structurally similar to the natural substrates. As isoleucine,
leucine and valine are primarily recognised by size...[Show more] exclusion,
all isosteres with a methyl group replaced by similarly-sized
chlorine are mistaken as substrates, and the ability of halogens
to mimic the bonding interactions of sulfur enabled the design of
a chlorinated and a brominated analogue of methionine.
Amino acids with chlorine at the 4-position, incorporated into
proteins in place of isoleucine, leucine or methionine during
cell-free protein expression, were found to trigger cleavage of
the proximal peptide bond on the C-terminal side at elevated
temperatures. The reaction mechanism is similar to that of
cyanogen bromide induced cleavage, driven by the formation of a
highly favourable 5-membered ring. This presents the first case
of heat induced proteolysis, resulting in almost instantaneous
peptide bond cleavage through exposure to 100 degrees Celsius in
water, and avoids the need for toxic, expensive or sensitive
external agents. When encoded in a fusion protein, the
chlorinated residues can be used for rapid separation of the
fusion partners.
The utility of the method was demonstrated through the
preparation of small peptides human gastrin releasing peptide
prohormone, cholecystokinin prohormone and oxytocin, lacking
isoleucine, leucine and methionine, respectively, expressed as
fusion proteins. Through simultaneous replacement of two amino
acids, deuterated and fluorinated analogues of the peptides were
also prepared. The method was then expanded to prepare more
complicated targets. Homologous substitution of leucine with
isoleucine, and vice versa, enabled the preparation of a small
protein aprotinin. A crystal structure of the mutated aprotinin
demonstrated that the protein structure was not affected by the
substitution, thus establishing that interchange of similar amino
acids can be used to overcome sequence limitations.
In stark contrast, amino acids with chlorine at the 3-position
are resistant to high temperature. Through the ability to
substitute valine or isoleucine during protein expression, the
chlorides were incorporated into aprotinin, thus establishing a
method for the preparation of proteins of essentially unlimited
size containing 3-chloro amino acids. Crystal structures of the
chlorinated aprotinin showed that the amino acid analogues are
not inherently detrimental to protein structure and can lead to
stable proteins, while the preparation through the expression of
heat-labile fusion proteins juxtaposed the differences in
reactivity between amino acids halogenated at the 3- and
4-positions.
Items in Open Research are protected by copyright, with all rights reserved, unless otherwise indicated.