This document discusses the recombinant expression of the Borrelia burgdorferi porin P13 in different expression systems. Key points: - P13 was cloned into expression vectors pARAJS2 and pICH31160 and expressed in E. coli and Nicotiana benthamiana plants respectively. - Expression was confirmed via SDS-PAGE and western blot, showing successful high yield production of recombinant P13 protein in both systems. - Recombinant expression of P13 will allow large scale production for development of new diagnostic and therapeutic strategies, and further study of P13's structure and function in Borrelia burgdorferi.

1. P13, an atypical porin of Borrelia
burgdorferi.
Dr. Iván Bárcena Uribarri
University of Wuerzburg
Wuppertal, April 8-10, 2011

2. Contents
Introduction
The P13 porin in Borrelia burgdorferi
Results and conclusions
Cloning of P13 in E. coli and Nicotiana benthamiana

3. Introduction
Borrelia P13 porin P13
TMHMM server v 2.0
computer prediction
Rosa PA. et al – 2005 – Nat. Rev. Microbiol.

4. Introduction
The general trimeric porin model versus P13.
P13 of Borrelia
16-18 β-strands
Examples : OmpF (diffusion porin)
LamB (specific porin)

5. Introduction
The general trimeric porin model versus P13.
16-18 β-strands
P13 of Borrelia
Examples : OmpF (diffusion porin)
LamB (specific porin)

6. Introduction
P13 periplasmic C-terminus processing
n monomers ?
BAM OM
? CtpA
OmpC
c-terminus P13
Sec IM
OmpC Signal seq. c-terminus P13 Signal seq.

7. Introduction
P13 paralogues*
Gene Nucleotide position Location Function
bb0034 32089–31553 Chromosome Channel-forming protein P13
bba01 588–1070 lp54 Channel-forming protein BBA01**
Conserved hypothetical protein,
bbg03 2104–2492 p28-2
authentic frameshift
bbh41 28197–27628 lp28-3 Conserved hypothetical protein
bbi31 20127–19618 lp28-4 Conserved hypothetical protein
Conserved hypothetical protein,
bbj02.1 1475–2367 lp38
pseudogene
Hypothetical protein (located inside
bbj03 1593–1742 lp38
BBJ02.1)
bbq06 3623–4105 lp56 Conserved hypothetical protein
Conserved hypothetical protein,
bbq81 49246–49047 lp56
pseudogene
*Pinne M. et al (2004) Microbiology.
**Pinne M. et al (2006) J Bacteriol.

8. Introduction
P13 paralogues*
P13
P13 Homologues
Chaconas G. 2005
Mol. Microbiol.
Why are so many copies of the P13 gene in these genetically reduced bacteria?

9. Introduction
P13 immunogenic potential
-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.
Pinne M. et al (2004) Microbiology.

10. Recombinant obtaining of P13
from B. burgdorferi in different
expression systems

11. Results
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

12. Results
DNA constructs
To transform E. coli: To transform A. tumefaciens:
pARAJS2 pICH31160
vector vector
Potato virus X
Escherichia coli BL21 Omp8 rosetta Agrobacterium tumefaciens

13. Results
Expression in Tobacco plants Plant cell
DNA virus sequence
Transcription
Nucleus
Agroinfiltration of
Nicotiana benthamiana RNA virus sequence
Export
RNA virus sequence
viral replication
Cytoplasm Cell-to-cell and/or
Agrobacterium strain
systemic movement
carrying viral vector
translation
protein procesing
Adapted from Marillonnet, S. et al. (2004) Proc Natl Acad Sci U S A.

14. Results
rP13 expression in Nicotiana benthamiana
SDS-PAGE Western Blot

15. Conclusions
P13
• 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

16. Thank you for listening !