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P13, an atypical porin of Borrelia burgdorferi.
Dr. Iván Bárcena UribarriUniversity of Wuerzburg
Wuppertal, April 8-10, 2011
Introduction
The P13 porin in Borrelia burgdorferi
Results and conclusions
Cloning of P13 in E. coli and Nicotiana benthamiana
Contents
Borrelia P13 porin
Rosa PA. et al – 2005 – Nat. Rev. Microbiol.
Introduction
TMHMM server v 2.0computer prediction
P13
The general trimeric porin model versus P13.
Examples : OmpF (diffusion porin)LamB (specific porin)
16-18 β-strands
Introduction
P13 of Borrelia
The general trimeric porin model versus P13.
Examples : OmpF (diffusion porin)LamB (specific porin)
16-18 β-strands
Introduction
P13 of Borrelia
P13 periplasmic C-terminus processing
Sec
BAM
CtpA
P13 Signal seq.c-terminus
P13c-terminus
Introduction
OmpC Signal seq.
OmpC
IM
OM
?
n monomers?
P13 paralogues*
Gene Nucleotide position Location Function
bb0034 32089–31553 Chromosome Channel-forming protein P13
bba01 588–1070 lp54 Channel-forming protein BBA01**
bbg03 2104–2492 p28-2Conserved hypothetical protein, authentic frameshift
bbh41 28197–27628 lp28-3 Conserved hypothetical protein
bbi31 20127–19618 lp28-4 Conserved hypothetical protein
bbj02.1 1475–2367 lp38Conserved hypothetical protein, pseudogene
bbj03 1593–1742 lp38Hypothetical protein (located inside BBJ02.1)
bbq06 3623–4105 lp56 Conserved hypothetical protein
bbq81 49246–49047 lp56Conserved hypothetical protein, pseudogene
*Pinne M. et al (2004) Microbiology.**Pinne M. et al (2006) J Bacteriol.
Introduction
P13 paralogues*
Chaconas G. 2005 Mol. Microbiol.
Why are so many copies of the P13 gene in these genetically reduced bacteria?
Introduction
P13
P13 Homologues
P13 immunogenic potential
Introduction
Pinne M. et al (2004) Microbiology.
-Outer-surface-epitope regions are the most heterogeneous.-Design of three fragments (ABC) and recombinant expression.- Antibody 15G6 that recognizes the natural epitope only recognized fragment B.
Recombinant obtaining of P13 from B. burgdorferi in different
expression systems
Recombinant expression of P13:
Why P13?:
- Development of new targets for diagnosis/therapie strategies.
- Knowledge about structure and function of P13 and its function in Borrelia.
Why recombinant and no native?:
- Production of large amounts of P13
- Reduction of the costs
- Reduction of the time
- Reduction of the infection risk
Results
Potato virus X
pARAJS2vector
pICH31160vector
Agrobacterium tumefaciensEscherichia coli BL21 Omp8 rosetta
Results
DNA constructs
To transform E. coli: To transform A. tumefaciens:
Cytoplasm
Nucleus
Agroinfiltration of
Nicotiana benthamiana
Agrobacterium strain
carrying viral vector
RNA virus sequence
viral replication
Transcription
Export
translation
protein procesing
Cell-to-cell and/or
systemic movement
Results
DNA virus sequence
RNA virus sequence
Expression in Tobacco plants Plant cell
Adapted from Marillonnet, S. et al. (2004) Proc Natl Acad Sci U S A.
Results
rP13 expression in Nicotiana benthamiana
SDS-PAGE Western Blot
Conclusions
• P13 was produced successfully in two systems with a high yield expression.
• Development of better diagnosis/treatment strategies.
• Better understanding of the biological function of this fascinating protein
P13
Thank you for listening !