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Rhagium inquisitor AFP (RiAFP) may overcome limitations of current AFPs
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Western Blot (α-6His)
RiAFP
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9.0mg/mL RiAFP
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Aaron Hakim Darren Zhu Chidi Akusobi
Durga Thakral Alexander Li Kara Brower
Aaron Lewis Steven Zhu Hill Ryan
References
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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
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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
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