BLBS102-c36 BLBS102-Simpson March 21, 2012 18:47 Trim: 276mm X 219mm Printer Name: Yet to Come
36 Biological Activities and Production of Marine-Derived Peptides 697Production of Peptides by Recombinant
DNA TechnologyTechnologies for heterologous protein expression have been in-
vented and developed exponentially (Terpe 2006). Although
there are a variety of expression systems by which a protein
or peptide can be recombinantly produced, expression of a pep-
tide in a recombinant host cell has met with limited success.
Frequently, peptides are poorly expressed in bacteria because in-
tracellularly the peptide may encounter one or more difficulties
including insolubility, instability, and degradation. If a peptide is
successfully produced recombinantly and purified, such isolated
peptide may have reduced biological activity compared to the
same amino acids that are part of the protein from which the
peptide sequence is derived. Such reduced activity is typical be-
cause the active domain of the peptide is conformational rather
than linear; and thus, in solution, the isolated peptide does not
occur in proper conformation for full biological activity, or such
proper conformation is only one of many alternative structures
of the peptide.
To overcome the difficulties encountered in expression of pep-
tides, there have been various attempts to express a peptide in
a conformation reflective of the conformation of the peptide
when expressed as part of the protein from which it is derived.
One of those approaches is to express the peptide as part of
a fusion protein (Liu et al. 2007). Typically, a fusion protein
consists of a microbial (e.g., bacterial) polypeptide backbone
into which is incorporated an amino acid sequence representing
one or more heterologous peptide sequences. A step in fusion
protein expression comprises inserting into a gene, encoding the
microbial polypeptide, a nucleic acid sequence encoding one
or more heterologous peptides. Usually, the gene is part of an
expression vector, such that when the vector is introduced into
a host cell system, the fusion protein is then produced either
as remaining host cell-associated, or secreted into the culture
medium of the expression system. In the art of recombinant
protein expression, there remains a need for new systems, and
new compositions for the production and delivery of biologi-
cally active and stable peptides for use. For example, necessary
control elements for efficient transcription and translation (pro-
moters and enhancer sequences) that are compatible with, and
recognized by the particular host system used for expression
must be incorporated. In the case that the fusion protein may
be lethal or detrimental to the host cells, the host cell strain/line
and expression vectors may be chosen such that the action of
the promoter is inhibited until specifically induced. A variety
of operons such as the trp operon, are under different control
mechanisms. The trp operon is induced when tryptophan is ab-
sent in the growth media. The PLpromoter can be induced by an
increase in temperature of host cells containing a temperature
sensitive lambda repressor. In this way, greater than 95% of the
promoter-directed transcription may be inhibited in uninduced
cells. Thus, expression of the fusion protein may be controlled
by culturing transformed or transfected cells under conditions
such that the promoter controlling the expression from the fu-
sion sequence encoding the fusion protein is not induced, and
when the cells reach a suitable density in the growth medium, thepromoter can be induced for expression from the inserted DNA.
Accordingly, a recombinant DNA molecule containing a fusion
sequence, can be ligated into an expression vector at a specific
site in relation to the vector’s promoter, control, and regulatory
elements so that when the recombinant vector is introduced into
the host cell, the fusion sequence can be expressed in the host
cell. The recombinant vector is then introduced into the appro-
priate bacterial host cells, and the host cells are selected, and
screened for those cells containing the recombinant vector. Se-
lection and screening may be accomplished by methods known,
depending on the vector and expression system used (E. coli,
B. subtilis, yeast, fungi, mammalian, and insect cells). Bacterial
expression is the usual starting point for expression of heterolo-
gous proteins. Bacterial fermentations are inexpensive and can
reach high cell densities resulting in high volumetric yields of the
target protein. The use ofB. subtilishas many advantages, such
as its GRAS status and easy and inexpensive culturing meth-
ods that can result in very high cell densities. Baculovirus/insect
cell systems have found wide application for the expression of
highly recalcitrant proteins such as protein tyrosine kinases. As
with bacterial expression systems, yeasts (Saccharomyces cere-
visiaeandPichia pastoris) offer relatively inexpensive growth
media and high-density fermentation. Furthermore, yeast offers
the capability of carrying out limited posttranslational modi-
fications such as disulfide bond formation and glycosylation.
Mammalian cell expression has become the system of choice
for production of complex, glycosylated biotherapeutic proteins
such as antibodies, growth factors, and fertility hormones. Sev-
eral developed methodologies appropriate for large-scale indus-
trial production of intact recombinant peptides in bacteria have
been developed without significant toxicity and a complicated
purification method. Using this technology, pilot-scale fermen-
tation can be performed to produce large quantities of biolog-
ically active peptides. Together, this new method represents a
cost-effective means, compared to chemical peptide synthesis to
enable commercial production of peptides in large-scale under
good laboratory manufacturing practice (GMP) for application
in humans.Membrane TechnologyMembrane processes involve separating components by means
of selective permeability through the membrane according to
flow under pressure over the surface of membrane. The technol-
ogy is actually a family of processes that include reverse osmo-
sis, nanofiltration, ultrafiltration, and microfiltration, which can
be differentiated by the separation range (Short 1995). Because
of the fact that the differences in physicochemical properties of
active sequences are often small, the specific separation of one
or more peptides from a raw hydrolysate is difficult. Until now,
ultrafiltration has provided the best method available for the en-
richment of peptides (Korhonen and Pihlanto 2006, Kozlov and
Moya 2007), but the selectivity between peptides of close sim-
ilar MW is poor. Among them, ultrafiltration has been widely
used to enrich bioactive peptides from protein hydrolysates by
which enzymatic hydrolysis can be performed through either