226 11 Small Functional Peptides and Their Application in Superfunctionalizing Proteins
α‐helical peptides being often more active than structurally less defined peptides
and peptides with the gamma‐core motif often being very active [110].
Due to their overall positive charge, antimicrobial peptides can accumulate at
the negatively charged microbial cell surface – which often contains acidic poly-
mers in Gram‐negative as well as Gram‐positive bacteria. After self‐mediated
uptake, they insert into the cytoplasmic membrane, disrupting its physical integ-
rity [111]. Some peptides can also cross the membrane and act on intracellular
targets [107]. Besides their high potential as broad‐range antibiotics, recent stud-
ies point toward a second function: the cationic peptides – which are not only
produced by bacteria in their fight to populate ecological niches but are also
found in higher organisms as defense mechanism [112] – are modulators of
innate immunity [113, 114]. This property is discussed to have potential for the
development of novel anti‐infective therapeutic strategies [115]. A comprehen-
sive database containing the sequences and properties of animal and plant pep-
tides is available [116].
To meet the needs of basic research and clinical trials, large quantities of
highly purified peptides are required. Although some peptide antibiotics are
synthesized non‐ribosomally by complex peptide synthetases, most of the pep-
tides are genetically encoded. Recombinant production in bacteria offers an
attractive approach for cost‐effective large‐scale peptide manufacture. A data-
base housing information on recombinant approaches to generate suitable
amount of antimicrobial peptides for biological and structural studies has been
established [117]. The field of antimicrobial peptide production therefore nicely
exemplifies the attempt to overcome shortcomings in the chemical production
of peptides – a field that could also be of great interest for the production of
mimotopes.
Most production approaches resemble the natural production mechanism of
antimicrobial peptides in their host [118]: to protect the production host from
peptide toxicity and the peptide from cellular degradation, the target peptides
are produced as larger precursors that are then processed by proteases to release
the actual active peptide moiety. Like this a variety of fusion partners and release
strategies have been explored [119]. Besides host toxicity and degradation, also
the intrinsic hydrophobicity of the peptides impairs its soluble production when
overproduced in a bacterial host. Commonly used strategies involve solubility‐
enhancing fusion partners like thioredoxin and glutathione‐S‐transferase (GST)
[120], but also small aggregation‐promoting carriers, for example, PurF [121] or
ketosteroid isomerase [122], were explored. The rationale for the latter is to
channel the peptide fusion into inclusion bodies to circumvent host toxicity and
degradation while still having easy access to the peptide. Release from a carrier
can be achieved by chemical hydrolysis or by specific proteases like TEV
protease [119].
However, current yields for purified peptides are limited to milligrams per liter
culture and current efforts focus on finding novel scaffolds for efficient expres-
sion. Again to the best knowledge of this author, peptide production using per-
missive site within solubilizing scaffold has not been explored yet, but seems to
be a promising alternative to current approaches, especially to address the prob-
lem of peptide degradation during production.