Development of an Anchoring System for Protein Display on the Cell Wall Surface of Lactococcus Lactis Mg1363
Lactococcus is one of the lactic acid bacteria that are widely used in various food and fermentation processes. They have been used for many centuries in food fermentation processes and are considered as GRAS organisms that can safely be used in medical and veterinarian applications. The anchorin...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2006
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/5982/1/FBSB_2006_9.pdf |
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| Summary: | Lactococcus is one of the lactic acid bacteria that are widely used in various food and
fermentation processes. They have been used for many centuries in food fermentation
processes and are considered as GRAS organisms that can safely be used in medical
and veterinarian applications. The anchoring of proteins to the cell surface of
Lactococcus using recombinant DNA techniques is an exciting and emerging research
area that holds great promise for a wide variety of biotechnological applications.
Presently available anchoring systems are based on recombinant bacteria displaying
proteins or peptides on the cell surface. The objectives of this study are to develop
surface display vectors and study the display of recombinant proteins on the surface of
Lactococcus lactis.
Several anchor proteins have been identified in L. lactis. In this study the gene coding
for the cell wall binding domain of L. lactis cell wall anchor proteins AcmA and NisP
were amplified by PCR and cloned into an E. coli expression vector. Sequencing results
showed 98% homology to published sequences. The plasmids designated as pSVacrn
and pSVnp were then transformed into E. coli where SDS-PAGE and Western blot
analyses showed that the cell wall binding domain of acmA and nisP genes were
successfblly expressed at the expected sizes 15 kDa and 18 kDa respectively. After
mixing of the purified recombinant AcrnA and NisP proteins with L. Iactis cells, their
presence on the bacteria cell surface was observed by whole cell ELISA, ~ i ~ ~ b i n d i n g
and fluorescence microscopy analysis.
The stability assay indicates that the binding of AcrnA protein to the lactococcal cell
surface was stable and can be retained on the cell wall surface for at least 5 days. The
results form the pH study indicatedthat low pH had no significant effect on the stability
of bound His-tag AcmA protein. Whilst the cell wall binding domain of AcmA was
shown to be able to anchor to the cell surface of other Gram-positive bacteria tested in
this study, AcrnA protein was not able to bind to the surface of E. coli (Gram-negative)
cells. Studies were also carried out to enhance the binding of AcmA protein to L. lactis
cells where pretreatment of L. lactis with 10% TCA was shown to improve binding of
the AcmA protein.
The new method developed for cell surface display of recombinant proteins on L. lactis
was evaluated for expression and display of foreign proteins. The gene coding for the
N-terminal epitope regions (VP11-67aaa nd VP13s-looaao) f VPl protein of Enterovirus 71
(EV71) were subcloned upstream to the cell wall binding domains sequences of
plasmids pSVacm and pSVnp. SDS-PAGE and Western blot results confirmed the
expression of N-terminal regions of VP1 protein as AcmA and NisP fusion proteins in
E. coli. Wholecell ELISA and imrnunofluorescence microscopy assays showed the
successfbl display of VPl protein of EV7 1 on the surface of L. lactis. The success of
docking VP 1 1-67aa and VP 1 35-100aa epitopes of VP 1 on the surface of L. lactis cells using
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the anchoring system developed in this study, open up the possibilities of peptide and
protein display for not only Lactococcus but of other Gram-positive bacteria.
Preliminary studies showed that mice immunized with L. Iactis displaying VP1147, or
VP13s-looafau sion proteins were able to induce an immune response against the VP11-
6 7 o~r V P135-100a(aa ntigens). The new method developed for surface display has the
potential to a variety of applications including screening of polypeptide libraries,
development of live vaccines, construction of whole cell allosteric biosensors, and
signal bansduction studies. |
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