Nature’s Antifreeze:Microbial de-icing solutions for ice cream,

aircraft and organ transport

Yale iGEM 2011

Challenge:

• Achieve large scale, stable extracellular production of highly active antifreeze protein (AFP) using Escherichia coli.

• Express, characterize, and purify:– Zoarces elongatus ZeAFP AFP (ZeAFP)– Tenebrio molitor AFP (TmAFP)– A novel, hyperactive Rhagium Inquisitor AFP

(RiAFP)

AFPs

Applications

Motivation

• Previous iGEM project on eGFP-TmAFP failed to characterize expression

• Current AFPs used in industrial applications are far less active than more recently isolated AFPs

• Current cost of type I/II/III anti-freeze proteins:– $10,000 / gram– http://www.afprotein.com/products.htm

RiAFP• Cerambycid beetle Rhagium inquisitor is v. cold-tolerant

– Hibernates in polar regions– Readily super-cools to -20oC to -30oC– Expresses RiAFP in hemolymph and tissues

• Previous studies (Kristiansen et al., 2008 and 2010) have: – isolated protein from hemolymph, – measured anti-freeze activity (TH), – salt enhances anti-freeze activity– determined primary sequence of RiAFP,

Advantages of using RiAFPRiAFP

Antifreeze activity as a function of concentration of various insect AFPs.(Kristiansen, et al 2010)

Insect AFPs are up to 100x more active than fish AFPs at low temperatures(Tong, et al 2008)

TmAFP

SbAFP ISbAFP II

Advantages of using RiAFP

• Single disulfide bond (less tightly folded compared to other active insect AFPs)

• Small protein (136 amino acid; 12kDa) • Novel AFP

Summary of AFPsAFP Source Size Disulfide

BondsMax.Activity

Structure

ZeAFP Zoarces elongatus

14 kDa 0 1 °C Determined

TmAFP Tenebrio molitor

8.4 kDa 8 5.5 °C Determined

RiAFP Rhagium inquisitor

12.8 kDa 1 7 °C Undetermined, no homology

Schematic Presentation of Expression Constructs

PelB eGFP

TEV

RiAFP His6

SS RiAFP His6

ZeAFP His6PelB

His6TmAFPeGFP

Generously provided by Peter Davies, Queens University

Generously provided by Team Tokyo Tech, 2009

SS eGFP

TEV

RiAFP His6

Generously provided by IDT

Plasmid Construct

• All constructs will be in pSB1K3– High copy, Kan resistance

SpeI Not I PstIEcoRI Not I XbaI

Characterization

• Basic Characterization:– GFP Fluorescence Assay– SDS/Page (supernatant and pellet)– Western blot (His6 antibody)

Assays – Fikrig Lab

• Freeze tolerance survival assay– Induce protein expression in E. coli– Harvest cells, wash in ddH2O, resuspend ddH2O

– Plate cells, assay viability, then freeze & thaw tube and assay viability once more

• Modified growth assays– Comparison of growth at 4oC induced/uninduced

Assays – Fikrig Lab

• Ice recrystallization assay (Graham et al, 2009)

Buffer DenaturedBuffer + I. scapularis AGFP

Buffer + ZeAFP AFP

Assays – Fikrig Lab

• Ice morphology assay (DeLuca, et al 1998)

(a) in the absence of protein (b) in the presence of thioredoxin

(10 mg/ml) in 100 mM NH4HCO3 (pH 7.9)

(c) in the presence of maltose-binding protein (20 mg/ml) in 100 mM NH4HCO3 (pH 7.9).

Right panel: 100 mM NH4HCO3 (pH 7.9) and mM concentrations:

(d) ZeAFP AFP, (e) TRX: ZeAFPAFP,(f) MBP: ZeAFPAFP

(1) Achieve stable, high-level expression of ZeAFP and TmAFP

• PCR out ZeAFP from pET20b expression vector clone into iGEM plasmid backbone and submit as biobrick

• Induce expression of ZeAFP (on pET20b) in BL21(DE3)

• Characterize ZeAFP by SDS/PAGE and/or western– Supernatant/Pellet

• Practice survival assays and ice assay• Purification?

PelB ZeAFP His6T7 –IPTG TerminatorRBS

ZeAFPPelB His6RBS

(1) Achieve stable, high-level expression of ZeAFP and TmAFP

• Transform eGFP-TmAFP-His6 fusion biobrick (Tokyo tech, 2009) into Origami and Bl21 cells

• Express at different temperatures• Characterize expression by fluorescence /

SDS / Western• Survival Assay• Ice Assay• Purification?

(2) Achieve stable, high-level, extracellular expression of RiAFP

• Construct following bricks:

PelB eGFP

TEV

RiAFP His6 TerminatorRBS

PelB eGFP

TEV

RiAFP His6T7 –IPTG Terminator

SS eGFP

TEV

RiAFP His6Promoter-RBS Terminator

RiAFP His6

PelB RiAFP His6T7 –IPTG Terminator

Promoter-RBS Terminator

RBS

RBS

(2) Achieve stable, high-level, extracellular expression of RiAFP

• Characterize expression by fluorescence / SDS / Western

• Survival Assay• Ice Assay• Comparison of activity of RiAFP versus eGFP-

RiAFP fusion protein – cooperativity?• Purification?

Schematic representation of cooperativity and non-cooperativity

• Previous studies suggest that ZeAFP and TmAFP function independently (no cooperativity)– Fusion proteins were

slightly more active than the free ZeAFP AFPs (DeLuca, et al 1998)

• Other studies on AFGP and type I, ZeAFP AFP from fish have displayed cooperativity (Ben, et al 2006)

Timeline• What we have accomplished thus far:

– Received BioBrick library, stabs of ZeAFP AFP and eGFP-TmTHP– Ordered RiAFP gene from IDT DNA (many thanks!)– Minipreps of eGFP-TmAFP, ZeAFP antifreeze, promoter bricks, GFP bricks,

terminator bricks, RBS bricks, OmpA, PelB leader sequences– Made Competent DH5-alpha cells– Design of primers to transfer ZeAFP into iGEM standard vector– Practice E. coli survival assay

• June:– Preparation of Origami competent cells– PCR out ZeAFP AFP from pET20b vector & ligate into standard iGEM vector– Express and purify eGFP-Tm-THP in Origami and BL21 cells

• Fluorescence/SDS/Western– Ice assays for positive controls

Timeline

• July:– Clone synthetic R. inquisitor gene into standard iGEM

vector– Ligate to various promoters/etc– PCR out RiAFP and clone into standard iGEM vector– Complete ice assays

• August: – Spillover– Optimize export quantities using MAGE technique

Important Dates

• 6/7/11 Project Defense• 10/8/11 - 10/10/11 iGEM 2011 Regional

Jamboree: Americas• 11/5/11 - 11/7/11 iGEM 2011 World

Championship Jamboree, MIT

Thank you!• Raymond and Beverly Sackler Institute for Biological,

Physical and Engineering Sciences• Yale College Dean’s Office• Professor Farren Isaacs• Professor Nigel Grindley• Graduate Student Advisors• Integrated DNA Technology• W.M. Keck Foundation• Professor Peter Davies• Tokyo Tech 2009• iGEM

References• Bouvet, V.R., Lorello, G.R. & Ben, R.N. Aggregation of Antifreeze Glycoprotein

Fraction 8 and Its Effect on Antifreeze Activity. Biomacromolecules 7, 565-571 (2006).

• DeLuca, C.I., Comley, R. & Davies, P.L. Antifreeze proteins bind independently to ice. Biophys J 74, 1502-1508 (1998).

• Garnham, C.P. et al. Compound Ice-Binding Site of an Antifreeze Protein Revealed by Mutagenesis and Fluorescent Tagging. Biochemistry 49, 9063-9071 (2010).

• Bar, M., Bar-Ziv, R., Scherf, T. & Fass, D. Efficient production of a folded and functional, highly disulfide-bonded [beta]-helix antifreeze protein in bacteria. Protein Expression and Purification 48, 243-252 (2006).

• Kristiansen, E., Pedersen, S.A. & Zachariassen, K.E. Salt-induced enhancement of antifreeze protein activity: A salting-out effect. Cryobiology 57, 122-129 (2008).

• Kristiansen, E. et al. Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor. Insect Biochemistry and Molecular Biology 41, 109-117 (2011).

Questions?

Extra slides

AFPs depress the freezing T of fish blood several hundred times more efficiently than regular salts

Structure/Mechanism of AFPs

• Structures have evolved independently

• Activity varies from about 1 °C in blood of polar to 5–7 °C in the hemolymph of many overwintering insects

• Various mechanisms have been proposed to explain the activity/interaction between AFPs and ice. – Adsorption-inhibition hypothesis : Gibbs –Thompson effect – Ice binding surfaces are relatively hydrophobic

Previous Studies

• Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor, Insect Biochemistry and Molecular Biology, 2010– Report on primary structure of a 12.8 kDa AFP from this

beetle (RiAFP). – It has a high capacity to evoke antifreeze activity as

compared to other known insect AFPs and it is structurally unique in several aspects.

– Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT

Previous Studies• Salt-induced enhancement of antifreeze protein activity: a

salting-out effect, Cryobiology, 2008– Report on salt-induced enhancement of the antifreeze activity

of an antifreeze protein from the longhorn beetle– Activity is predetermined by a steady-state distribution of the

AFP between the solution and the ice surface region• Isolation and characterization of hemolymph antifreeze proteins

from larvae of the longhorn beetle Rhagium inquisitor (L), Comp. Biochem. Physiol, 2005– Procedure to isolate AFPs from hemolymph of larvae and

presents some characteristics of structure – 22% ala, 20% glyc, 34% threonine, 17% serine, 9% Glyx– Mass, pH stability (3-12)

Justification for fusion constructs• Signal sequence:

– More easily purified from culture media– Greatly facilitates downstream processing– Continuous cultures for large-scale production– Potential toxicity for cell? – “Sec SRP TAT” (disulfide bond, Sec or SRP; PelB common)

• eGFP: – Flourescence measurements– Improved solubility– Improved staining with Comassie brilliant blue

• TEV cleavage: – Easily purified; co-operativity analysis.

• His6 Tag