Upload
ian-martin
View
246
Download
1
Embed Size (px)
Citation preview
“Assessing the Sweet Tooth of a Fungal Lectin”
Student Name: Ian MartinStudent ID: 11532343 Supervisor : Dr. Brendan O’Connor
What Are Lectins ?Definition : Lectins are Carbohydrate Binding Proteins (CBP) that recognize and bind to specific glycans in a non-catalytic and reversible manner.
Roles and potential applications of fungal lectins.
Enyme-Linked
Lectin Assay ELLA
What is AAL-2 ?
Amino Acid Sequence of PVL
Psathyrella Velutina Lectin
(PVL) is 60% identical with
AAL-2
• Fungal Lectin from Agrocybe Aegerita Mushroom
• AAL-2 coding sequence (1224 bp)• Protein Size 43kDa• Binds to terminal N-acetylgucosamine
(GlcNac)• Induces Apoptosis in Certain Cancers• O-GlyNacylation Detection Device
Aims & Objectives1. Introduce mutations at GlcNac binding pockets of
AAL-2 using site directed mutagenesis .
2. Identity by 15 % SDS-Page Gel analysis an efficient E.coli expresser which can produce high levels of recombinant AAL-2.
3. Selectively purify AAL-2 using IMAC purification.
4. Characterise the binding efficiency of mutated AAL-2 using ELLA analysis. Mutated vs Non Mutated AAL-2
DNA Manipulation
Plasmid Isolation showed on a 0.7% Agarose SYBR stained Gel. DNA prep loaded in lane 7. MW used in lane 1 & 1 0.
Plasmid Isolation
Site Directed Mutagenesis
Dpn 1 Digestion
T4 Ligation
Transformation (JM109)
Site Directed Mutagenesis Products loaded onto 0.7% TAE Agarose SYBR stained gel . Lanes 2-5 PCR Products. MW in lanes 1 and 5
0.7 % Agarose SYBR stained gel containing digested DNA PCR products. Lanes 3 and 5 contain Digestions.Lane 1 loaded with MW.
Amp+ LB agar plate selecting JM109 transformants. 12 shiny circular colonies detected using concentrated JM109 prep.
Mutations To be Introduced
G54A G55A W56AExperimental
Setup
0.7 % Agarose SYBR stained Gel containing JM109 ( Lanes 2-5) and KRX PCR products (7-10). MW are in lane 6.
Wild Type Amino Acid Sequence D-A-G-G-W-224
Mutations To be Introduced G221A G222A W223A
DNA Manipulation
Sequence Results
JM109 KRXPlasmid Isolation
Site Directed Mutagenesis
Dpn1 Digestion
T4 Ligation
Transformation (JM109)
Protein Expression & Purification
Lane 11:Reference AAL , Lanes 12 and 13: JM109 protein samples. Lanes 1 and
14: MW
15 % SDS Page Protein Expression Check
15 % SDS-PAGE gel stained with Coomassie blue. Lanes 3 to 14 contain
no purified AAL-2. Lane 2: MW
Purified IMAC Samples
Troubleshooting Protein Purification 15% SDS-PAGE Lysate
Analysis
15% SDS-PAGE gel stained with Coomassie blue displaying lane 9 Filtered Lysate , lane 3 AAL-2 reference and MW
lane 2.
15% SDS-PAGE Analysis Fractions Unbound & Washes
15% SDS-PAGE gel stained with Coomassie blue showing fractions lanes
2-12 ,unbound lane 13 and 20Mm imidazole wash.
Troubleshooting15 % SDS-PAGE Protein Expression Check
15% SDS-PAGE gel showing expression levels of JM109 transformants (lanes 1-5), KRX transformants (Lanes 6-8) and old JM109 transformants (lanes 9-10) MW in lanes 5 and 12.
10
15 % SDS-PAGE IMAC Purified Samples
Coomassie blue stained 15% SDS-PAGE showing Filtered lysate (Lane 2) Unbound fraction (Lane 3) , Imidazole Washes 20-80 Mm (Lanes 4-6 ) and eluted fraction Lanes (8-13). MW are in (Lane 7)
Future Perspectives
ELLA Characterisation ?
Additional Purification Steps ?
Mutate An Additional Site ?
Conclusion • Successfully introduced silence mutations at GlcNac binding
pockets of AAL-2 using site directed mutagenesis .
• Identified by SDS gel analysis an efficient E.coli expresser which can produce high levels of recombinant AAL-2.
• Selectively purified AAL-2 using IMAC purification.
Future work
• Characterise the binding efficiency of mutated AAL-2 in respect to its wild-type using ELLA analysis.
Acknowledgements Dr. Brendan O’ConnorJonathan Cawley Donal Monaghan Conor Akintola Ciaran Greene Melissa Burke Alex Newitt
“ Success is a journey, not a
destination. The doing is often
more important than the outcome.
”
Arthur Ashe
Questions?