–

–

–

Rhagium inquisitor AFP (RiAFP) may overcome limitations of current AFPs

–

–

–

•

–

•

•

- + - +

Western Blot (α-6His)

RiAFP

•

•

•

a

••

9.0mg/mL RiAFP

•

–

•

1

2

3

4

5

•

•

•

•

•–

Aaron Hakim Darren Zhu Chidi Akusobi

Durga Thakral Alexander Li Kara Brower

Aaron Lewis Steven Zhu Hill Ryan

References

•

•

•

•

•

•

Backup Slides

Figure 6: Fluorimetry was used to measure flourescence. Excitation

wavelength 488nm. Emission scan 500 to 650 nm

Figure 3: SDS-PAGE stained with Coomassie blue;

Uninduced eGFP-TmAFP (left), induced eGFP-TmAFP (right)

Figure 5: Western Blot, probed with

His-6 antibody;

Uninduced eGFP-TmAFP (left),

induced eGFP-TmAFP (right)

Figure 4: Western Blot, probed with

mouse α-GFP-IgG2a antibody.

Uninduced eGFP-TmAFP (left),

Induced eGFP-TmAFP (right)

Figure 8: Western Blot, probed with eGFP antibody.

Lane 0: ladder, Lane 1: uninduced eGFP-RiAFP in BL21, Lane 2: induced

eGFP-RiAFP in BL21, Lane 3: uninduced eGFP-RiAFP in Origami, Lane 4:

induced eGFP-RiAFP in Origami, Lane 5: uninduced RiAFP in BL21, Lane 6:

induced RiAFP in BL21, Lane 7: uninduced RiAFP in Origami, Lane 8:

induced RiAFP in Origami

RiAFP: First Hyperactive Antifreeze Protein Expressed and Purified in Large Quantities

150mg/L cells, (0.2mM) RiAFP-GFP

RiAFP-eGFP

eGFP & TEV

RiAFP

RiAFP Cell Lysate Inhibits Ice Recrystallization

30% sucrose + Pure Water (-ve control)

30% sucrose + Lysate Buffer 30% sucrose + RiAFP Cell Lysate

•50X

Ice Recrystallization Inhibition: Splat Assay

Figure 13: Splat assay shows

concentration dependent

inhibition of ice recrystallization.

Ice recrystallization was

allowed to take place for five

hours at -10oC before imaging.

Ice Recrystallization Inhibition:

Figure 14: Splat assay

shows concentration

dependent inhibition of ice

recrystallization. Ice

recrystallization was

allowed to take place for

twelve hours at -10oC

before imaging

•

2.0 M ammonium carboxylate 1.6 M NaH2PO4, 0.4 M KH2PO4

10X 20X

4.5mg/mL RiAFP 9.0mg/mL RiAFP

𝐾

𝑗((1 − 𝑀)𝑁)𝐾−𝑗(1 − (1 − 𝑀)𝑁)𝑗

𝐾

𝑗=0

Key: Red = 1 cycle, Green = 10 cycles, Blue = 30 cycles, Orange = 50 cycles, Purple = 90 cycles.

𝐾

𝑗((1 − 𝑀)𝑁)𝐾−𝑗(1 − (1 − 𝑀)𝑁)𝑗

𝐾

𝑗=0