1. Bioinformatics
Identified VlsE1 as top hit for potential ganglioside-binding protein.
VlsE1 localizes to the outer membrane of the cell.
Known to be involved in late-stage Lyme disease [4].
Although crystal structure is available, its function is unknown.
VlsE1 is found on linear plasmid-28.
 Required for pathogenesis for Lyme disease [5].
Identified possible ganglioside-binding motif [6].
Sub-cloning and Expression
Commercially synthesized VlsE1.
Sub-cloned into pET-28 to give N-terminal Histidine tag.
Expression Studies
 Modified media and concentration of IPTG parameters to produce the
best yield of soluble VlsE1.
Purification
 Trial purification with His-Select resin.
Drew E. Phelan, Christopher W. Reid
REFERENCES
1 Eicken, C., & Sharma, V. (2002). Crystal Structure of Lyme Disease Variable
Surface Antigen VlsE of Borrelia burgdorferi. V. Biol Chem. 277.
2 Garcia- Monco, J.C., Seidman, R.J., Benach, J.L., (1995). Experimental
immunization with Borrelia burgdorferi induces development of
antibodies to gangliosides. Infect Immun 63, 4130-4137.
3 Girschick, H., Morbach, H., Tappe, D. (2009). Treatment of Lyme borreliosis.
Arthritis Research and Therapy.
4 Polypeptides and Methods for the Specific Detection of Antibodies in
Patients with a Borrelia Infection. USSN 8,431,135 B2.
5 Lawrenz, M., Wooten, R., Norris, S. (2004). Effects of vlsE
Complementation on Infectivity of Borrelia burgdorferi Lacking the
Linear Plasmid lp28. Infect Immun.
6 Matsubara, T., Onishi, A., Sato, T. (2012). Carbohydrate Recognition by
Pentadecapeptide Ligands for a Series of Sialylated Oligosaccharides.
Bioorganic and Medicinal Chemistry Journal.
INTRODUCTION
The Center for Disease Control (CDC) estimates that there are
around 35,000 cases of Lyme disease (including unreported
cases) within the United States in recent years [1]. Lyme
disease is known to be caused by Borrelia burgdorferi, a
spirochete diderm without any LPS. Left undiagnosed, this
disease can run rampant throughout the body causing several
long-term neurological affects including myelitis and facial
nerve paralysis[1]. B. burgdorferi has been shown to bind to
gangliosides [2]. However the B. burgdorferi proteins
responsible have yet to be identified. The host-pathogen of the
Borellia infection process interaction has only been understood
in part. While B. burgdorferi is highly treatable with antibiotics,
if left undiagnosed, some can develop chronic persistent Lyme
disease [3]. Our research goal was to identify possible
ganglioside-binding proteins that are found within Borrelia
burgdorferi sensu lato.
MATERIALS & METHODS
RESULTS DISCUSSION
Bioinformatics
Identified top hit utilizing BLAST-p algorithm and narrowed
results down to one hit as shown in Table 1.
Literature mining showed that VlsE1 was shown to be
involved in late-stage Lyme disease.
Found possible ganglioside-binding motif within amino
acid sequence of VlsE1.
Sub-cloning and Expression
Cloned VlsE1 into pET-28.
Successfully expressed VlsE1 into BL21(DE3) pLlysS
expression strain.
Began concentration trials and identified .10 mM IPTG to be
slightly more optimal as shown in Figure 4.
Explored growth media for optimal protein expression
(utilized 2xYT, Super Broth, Terrific Broth and Luria Broth).
Began first purification attempt.
As shown in Panel C, VlsE1 remained insoluble.
Department of Science and Technology, Bryant University
1150 Douglas Turnpike, Smithfield RI 02917
Shiga Toxin
Figure 1: Flow chart of bioinformatics analysis.
Figure 2: Flow chart of sub-cloning and expression work of
VlsE1.
FUTURE WORK
• Continue to optimize expression and purification.
• Continue to optimize different conditions (including growth
media, IPTG concentration and incubation temperatures).
• Initiate a GST-fusion protein purification trial.
• Initiate experiments in order to identify the function of VlsE1.
• Screen VlsE1 for capacity to bind gangliosides utilizing
immuno- thin layer chromatography.
• Biochemically quantify the protein- ganglioside interaction of
VlsE1 utilizing hydrogen/ deuterium exchange MALDI MS (4,5).VlsE1
A
Table 1: Results of Bioinformatics methods.
B
289- R G M A K D G KF AV -299
GT1-b Motif- R X X A (F/W) X X S X ● (V/L)
C
Blast into sensu lato strain
1 2 3 4 5 6
1 2 3 4 5 6 7 8 9 10
D
ACKNOWLEDGEMENTS
Special thanks to Rhode Island INBRE SURF program for funding.
The Reid Research Group and The Glycomics Lab at Bryant University.
Special appreciation to Bryant University Academic Affairs for their support.
1 2 3 4 5 6 7 8 9 10
Bait
Protein
Borrelia
Match GI
Proposed
Name
Query
Cover
E- Value Identity
Effector
Results
PSORT
Results
Literature
Mining-
Found in
late stage
Lyme?
β-amyloid 503789484 Hypothetical
Protein
50% 2.7 52% None Cytoplasm No
β-amyloid 490932207 LysM
domain/M23
71% 3.1 38% None Cytoplasm No
β-amyloid 657248935 Peptidase
M23
71% 4.3 38% None Cytoplasm No
Shiga
Toxin
488739783 HD domain
Protein
35% .41 33% None Periplasmi
cmembran
e
No
Shiga
Toxin
501805000 Hypothetical
protein
84% 1.5 23% None Periplasmi
cmembran
e
No
Shiga
Toxin
3068754 Lipoprotein
OspE variant
84% 1.6 23% None Periplasmi
cmembran
e
No
Shiga
Toxin
186974253 VlsE1 29% 2.3 42% Sec
secreted
Bacterial
membrane
Yes
Panel A: Tertiary Structures shown above for both the bait protein, Shiga Toxin Subunit B
(1DM0), and the target protein, VlsE1 (1L8W).
Panel B: Coomassie SDS-Page Protein Gel of varying concentrations of IPTG added to
VlsE1. Lanes: 1) protein marker, 2) 0mM IPTG, 3) 0.1mM IPTG 4) 0.25mM IPTG, 5) 0.5mM
IPTG, 6) 1.0mM IPTG.
Panel C: Coomassie SDS-Page Protein Gel of expression study utilizing different growth
media. (1mM IPTG) Lanes: 1)protein marker 2) 2xYT Broth after Sonicated 3)Super Broth
after Sonicated 4) Terrific Broth after Sonicated 5) 2xYT Broth Lysate 6) Super Broth Lysate
7) Terrific Broth Lysate 8)2xYT Broth Centrifuge Pellet 9) Super Broth Centrifuge Pellet 10)
Terrific Broth Centrifuge Pellet
Panel D: Western Blot of growth media trials. Lanes: See Panel C.
Figure 4:
Figure 4: Amino Acid sequence fragment from VlsE1 with the ganglioside GT1-b motif below
highlighting exact matches in blue and conserved matches in green. (GT1-b is known to bind to
Borrelia burgdorferi.[2])