“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?