RECOMBINANT EXPRESSION AND PURIFICATION OF THE

TWO SUBUNITS OF THE HAGFISH SLIME INTERMEDIATE

FILAMENT PROTEINS

Guide Name: Dr Paul Young

Student Name:Shruthi Lakshmi Narasimhan

Introduction

• Most primitive, jawless vertebrates, that possess a skull without a

vertebral column, having been in existence for over 330 million

years (Subramanian et al., 2008).

• Marine craniates that discharge profuse amounts of slime, in to

resist attacks by predators (Bernards et al., 2014; Koch et al.,

1995).

• Slime released is composed of mucin and threads, that are

produced by 150 slime glands lined ventrolaterally along the

body of the animal (Bernards et al., 2014; Koch et al., 1995).

• Two cell types, Gland Mucus Cells (GMCs) and Gland Thread

Cells (GTCs), that form mucin and skeins respectively, are

responsible for the formation of hagfish slime (Fudge et al.,

2005; Spitzer et al., 1984).

Fig 1a: Hagfish produces copious amounts of slime, in the presence of sea water, upon agitation; Fig1b: Hagfish produces slime as a defence mechanism against gill-breathing predators (Fudge et al., 2014)

The Pacific hagfish (Eptatretus stoutii), seen in California. Photograph by Norbert Wu, Science Faction/Corbis

Review of Literature

Am J Physiol Cell Physiol. 2008

α-β Transition:

The structure of IFs in hagfish slime is composed

primarily of α helical coiled coils, which when

strained in water extends, leading to the formation

of β sheets (Pinto et al., 2014; Fudge et al., 2003).

Review of Literature

Applications:

•In textile industries, the use of synthetic polymers made of petroleum based products has been on the decline, to be replaced by fibres made of renewable and non-toxic substances (Fudge et al., 2010).•Hagfish slime fibres prove to be sustainable in both production and disposal, with impressive mechanical properties.•Biomimetic materials that can be dissolved in formic acid and drawn into fibres after being cast in electrolyte buffer (Negishi et al., 2012).

α βα helix undergoes conformational shift to form β

1. Testing co-expression of IFα and IFγ in bacterial vector systems

2. Scaling-up recombinant expression of IFα and IFγ subunits from hagfish

slime intermediate filaments , using optimized parameters

3. Characterization of purification strategies to concentrate IFα and Ifγ

subunits of hagfish slime intermediate filaments

4. Formation of a novel biopolymer with material science applications

Objectives

Work plan

Methodology

Induced protein expression with IPTGantibiotic

Transformed cells are incubated at 37°C overnight

Only colonies of E.Coli that have been transformed will grow on antibiotic plate

Transformation by Heat-shock

SDS Polyacrylamide Gel Electrophoresis performed to detect the presence of desired protein

Methodology

IF proteins are further concentrated by dialysis, where urea is removed from eluted protein samples

Hagfish slime IF proteins are purified using Nickel NTA beads

Purified IFα and IFγ proteins are combined to form biopolymer membrane by drop-casting

1. Testing expression of IF proteins in various vector sustems: pCDFα pCDFγ empty vector

Plasmid preparation : Restriction digestion with BamHI and HindIII

Protein expression in pCDF vector system

Protein expression in pRSF vector system

pRSFγ expressed

pRSFα expressed

Pre Post Pre Post 7702 Ind. Ind. Ind. Ind. pRSFα pRSFα pRSFγ pRSFγ

Pre Post Pre Post Ind. Ind. Ind. Ind. pRSFα pRSFα pRSFγ pRSFγ

66.4KDa

97.2KDa

116 KDa

158 KDa

Results

10,000bp

4,000bp

2,000bp

pCDFα expressedpCDFγ expressed

Post induction pCDF γ Pre-ind Post induction pCDF α Pre ind M pCDF γ pCDF α

IFα IFα IFγIFαIFγIFγ

Basic protein

Acidic protein

IFα IFγ

7% Urea PAGE

Results

P Pre Post Pre Post Pre Induction Post Induction pCDFγ and pRSFα 7702 Ind. Ind. Ind. Ind. pCDFγ and (Colonies 1 to 5) pCDFα pCDFα pCDFγ pCDFγ pRSFα

66.4 KDa

97.2 KDa

116 KDa

158 KDa

Co-expression pCDFγ + pRSFα

P Pre Pre Post Pre Post Pre Induction Post Induction pRSFγ and pCDFα 7702 Ind Ind. Ind. Ind. Ind. pRSFγ and (Colonies 6 to 10) pCDFα pCDFα pCDFα pCDFγ pCDFγ pCDFα

66.4 KDa

97.2 KDa

116 KDa

158 KDa

Co-expression pRSFγ + pCDFαpCDF IF-α expressed pCDF IF-γ expressed

66.4 KDa

P7702 Pre Post Pre Post Induction pCDFα Induction pCDFα Induction pCDFγ Induction pCDFγ

2. Scaling-up recombinant

expression of IFα and IFγ subunits

from hagfish slime

intermediate filaments , using

optimized parameters:

3b)Batch purification (pCDFα)

3. Protein purification strategies:

P7702 Elutions 1 to 5 pCDFα Elution 1 to 5 pCDFγ

66.4 KDa

3a)Continuous purification

1 2 3 4 5 1 2 3 4 566.4 KDa

P7702 Pre Post Pellet Flow through Elution Elution1 Elution2 Ind Ind SolB SolC SolC

Purified Protein

Results

3c) Dialysis

Results

Dialysis at pH5.5

P7702 α γ α+γ α γ α+ γ α γ α+ γ Input supernatant Pellet

66.4KDa

Dialysis at pH7.5

66.4KDa

P7702 α γ α+γ α γ α+ γ α γ α+ γ Input supernatant Pellet

66.4KDa

P7702 α γ α+γ α γ α+ γ α γ α+ γ Input supernatant Pellet

Dialysis at pH9.5

A B C

D E F

Images A-C lyophilized natural hagfish filaments.

Images D-F polymerised synthetic hagfish filament .

4. Drop casting

Conclusion1. Although expression in both dual vector systems (pCDF and pRSF)

individually was successful, co-expression of IFα and IFγ proved to be unsuccessful.

2. Analysis of proteins in Urea PAGE showed that IFγ is acidic while IFα is the basic keratin-like IF protein counterpart.

3. Scaling-up production of Intermediate filament proteins was efficient, under optimized parameters.

4. Purification of proteins by nickel column chromatography, obtained upon scaling-up, proved to effective in extracting most of the protein.

5. In order to model a biopolymer membrane 5% weight/volume of protein is essential, for which dialysis of purified protein proved to be efficient. Dialysis at different pH presented similar results, so all future experiments involving dialysis were carried forward at pH7.5.

REFERENCES1. Negishi, Atsuko, et al. "The production of fibers and films from solubilized hagfish slime thread

proteins." Biomacromolecules 13.11 (2012): 3475-3482.2. Fudge, D. S., Gardner, K. H., Forsyth, V. T., Riekel, C. & Gosline, J. M. (2003). The mechanical properties of hydrated

intermediate filaments: insights from hagfish slime threads. Biophys. J. 85, 2015–20273. Hearle, John WS. "Protein fibers: structural mechanics and future opportunities." Journal of materials science 42.19

(2007): 8010-8019.4. Fudge, Douglas S., et al. "Hagfish slime threads as a biomimetic model for high performance protein

fibres." Bioinspiration & biomimetics 5.3 (2010): 035002.Ip, W.; Hartzer, M. K.; Pang, Y. Y.; Robson, R. M. J. Mol. Biol. 1985, 183, 365−375.

5. Subramanian, S., N. W. Ross, and S. L. MacKinnon. "Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.)." Fish & shellfish immunology 25.5 (2008): 625-632.

6. Bernards, Mark A., et al. "Spontaneous unraveling of hagfish slime thread skeins is mediated by a seawater-soluble protein adhesive." The Journal of experimental biology 217.8 (2014): 1263-1268.

7. Koch, E. A., Spitzer, R. H., Pithawalla, R. B., Castillos 3rd, F. A. & Parry, D. A. 1995 Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments. Int. J. Biol. Macromol. 17, 283–292

8. Fudge, Douglas S., et al. "Composition, morphology and mechanics of hagfish slime." Journal of experimental biology 208.24 (2005): 4613-4625.

9. Fudge, Douglas S., Sarah Schorno, and Shannon Ferraro. "Physiology, Biomechanics, and Biomimetics of Hagfish Slime." Annual Review of Biochemistry 0 (2014).

10. Downing, Stephen W., et al. "The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions." The Journal of cell biology 98.2 (1984): 653-669.