Fundamental Food Microbiology, Third Edition - Fuad Fathir

GENETICS OF SOME BENEFICIAL TRAITS 165
molecules for use as pharmaceuticals (immunity protein vaccine) and in agriculture
(insecticides). Genes of many heterologous proteins have been cloned in Lac. lactis
strains, such as chicken egg lysozyme, bovine prochymosine, a-amylase, pediocin
(a bacteriocin), luciferase, interleukin 2, cholera toxin B, tetanus toxin C, and Cry
1A insect toxin. Many of the genes express at low to high levels, but a few do not
express to the level of detection. 8,11
1. Expression of Interleukin
A murine interleukin 2 (mIL2) expression vector, pL2MIL2, was constructed from
pLET2N (originally developed from pLET2, the lactococcal T7 polymerase-based
system). The plasmid, pL2MIL2, encodes the part of the mIL2 gene that codes for
the mature protein, fused with the lactococcal usp 45 secretory leader signal. Lac.
lactis MG 1820 was transformed with this plasmid. When grown in an appropriate
growth medium, the cells synthesized the USP 45-mIL2 fusion protein and secreted
the mature mIL2 into the growth medium. The purified mIL2 was found to be
biologically as active as the natural mIL2. By similar procedures, other murine
interleukins (such as mIL-6 and mIL-10), murine interferon, and human interleukin
have been produced by Lac. lactis. 13 Recent studies have shown that daily ingestion
of Lac. lactis that can produce biologically active mIL-10 interleukin has cured and
prevented enterocolitis in mice. 14
2. Drug-Delivery System
Many lactic acid bacteria are normally present in the gastrointestinal (GI) system
of humans and food animals and birds, and some of them have beneficial effects on
the health and well being of the hosts (see Chapter 15). Currently, studies are in
progress to anchor antigens of pathogens associated with enteric diseases in humans
on the cell wall of suitable lactic acid bacteria. The live cells then can be used as a
drug-delivery vehicle to deliver antigens to stimulate antibody production via the
digestive tract. One method used to anchor proteins is to use Lac. lactis Prt P, which
normally remains attached to the cell wall with its carboxy-terminal domain. The
effectiveness of the carboxy-terminal cell wall anchor of Staphylococcus aureus
protein A to the cell wall of Lac. lactis is also being investigated.
3. Production of Pediocin in Heterologous Hosts
The bacteriocin pediocin PA-1/AcH is produced by many strains of Ped. acidilactici.
The molecule is translated as a 62-amino-acid prepediocin from which the 18-aminoacid
leader peptide at the NH 2 terminal is enzymatically removed during membrane
translocation through the ABC transporter system (Chapter 16). The 44-amino-acid
pediocin is then released in the growth medium. The phenotype is plasmid linked
and the structural gene with three other genes is arranged in an operon system, and
the protein encoded by the last gene acts as the ABC transporter. The DNA fragment
with all four genes has been cloned in several Gram-negative vectors and in a shuttle
vector, and the host strains produce active pediocin molecules, generally at lower
concentrations.
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