Polyelectrolyte Fiber Assembly of Plant-Derived Spider Silk-like Proteins

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Polyelectrolyte Fiber Assembly of Plant Derived Spider Silk-Like Proteins Congyue Annie Peng, Julia A. Russo, Todd A. Lyda, and William Richard Marcotte Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01552 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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Biomacromolecules

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Polyelectrolyte Fiber Assembly of Plant-Derived

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Spider Silk-Like Proteins

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Congyue Annie Peng†, Julia Russo†,‡, Todd A. Lyda†,§, William R. Marcotte, Jr.†,*

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†

Department of Genetics and Biochemistry, 130 McGinty Court, Robert F. Poole Agricultural

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Center, Room 154, Clemson University, Clemson, SC 29634

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KEYWORDS: Spider Silk, Polyelectrolyte Complex Formation, Fiber

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ABSTRACT

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Spider dragline silk is a proteinaceous material that combines superior toughness and

11

biocompatibility, a promising bio-material. The distinct protein structure and the fiber formation

12

process contribute to the superior toughness of dragline silk. Previously, we have produced

13

recombinant spider silk-like proteins in transgenic tobacco that are readily purified from plant

14

extracts. The plant-derived spidroin-like proteins consisted of native major ampullate spidroin 1

15

or spidroin 2 N- and C-termini flanking 8, 16, or 32 copies of their respective consensus block

16

repeats (mini-spidroins). Here, we present the generation of fibers from mini-spidroins

17

(rMaSp1R8 and rMaSp2R8) by polyelectrolyte complex formation using an anionic

18

polyelectrolyte, gellan gum. Mini-spidroins, when treated with acetic acid and crosslinked by

19

glutaraldehyde, formed a thin film at the interface when overlaid with a gellan gum solution.

20

Immediate pulling of the film resulted in autofluorescent fibrous materials from either mini-

21

spidroin alone or from a combination of rMaSp1R8 and rMaSp2R8 (70:30). Addition of chitosan

22

to the mini-spidroin solutions enabled continuous fiber production until the spinning dope supply

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Biomacromolecules

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was exhausted. When air-dried as-spun fibers were rehydrated and stretched in water, fiber

2

diameter decreased and overall toughness improved. This study showed that spider silk-like

3

fibers can be produced in large quantity through charge attraction which assembles chitosan,

4

mini-spidroins and gellan gum into fibrous complexes. We speculate that the spider silk self-

5

assembly process in the duct may involve attraction of variously charged chitinous polymers,

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spidroins and glycoproteins.

7 8

INTRODUCTION

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Spider silks are natural proteinaceous biomaterials, durable, strong and hypoallergenic. The

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major protein components of dragline silk and their properties have been reviewed

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extensively.1,2,3 Two proteins, Major ampullate Spidroin 1 (MaSp1) and Major ampullate

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Spidroin 2 (MaSp2), were found to be the predominant protein components in Nephila clavipes

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dragline silk.4,5 Based on a comparison of the original deduced cDNA sequences and amino acid

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sequence analysis of dragline silk, the ratio of MaSp1 to MaSp2 was estimated to be 3:2.4 More

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recently, Guehrs et al. (2008) estimated the amounts of MaSp1 to be 5-10 times higher than

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MaSp2 in solubilized fibers and gland contents.6 They also demonstrated that the ratio of MaSp1

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to MaSp2 was dependent on nutritional status, wherein starved spiders had decreased levels of

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MaSp2 compared to well-fed spiders. This observation demonstrates that fibers can be

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successfully made with different ratios of MaSp1 to MaSp2 and supports the suggestion that

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recombinant spidroins should provide a direct approach to generate designer fibers with desired

21

mechanical properties.

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Major ampullate spidroins are large proteins (250-350 kDa) composed of roughly 100 non-

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perfect block repeats as a large central domain flanked by short non-repetitive N- and C-terminal

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domains.7 The block repeat domain is largely hydrophobic and MaSp1 and MaSp2 spidroins


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Biomacromolecules

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have distinct repeat domains. For MaSp1, the consensus block repeat contains (GA)n, (GGX)n, or

2

(GX)n, where X is often A, Y, L, or Q, and poly-alanine.5,8,9,10 For MaSp2, the consensus block

3

repeat is proline rich (~15%) and contains (GPGXX)n, where X is usually Q, G or Y, and poly-

4

alanine.4 The poly-alanine motifs assemble into β-sheet crystals during fiber assembly and are

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the major components of the highly ordered fiber structure

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higher amounts of β-sheet crystal percentage displayed higher elasticity.14

11,12,13

Fibers characterized with

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The non-repetitive N- and C-terminal domains (NTD and CTD, respectively) are conserved

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among species15,16 and, unlike the repeat domain, most of the amino acids in the NTD and CTD

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have hydrophilic side chains, suggesting they may facilitate solubility at the high protein

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concentrations found in the storage sac of the gland. CTDs form homodimers through inter-

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molecular salt bridges and the monomers are covalently connected by a disulfide bond.17 The

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CTD plays an important role in β-sheet alignment during fiber formation.18,19 NTDs also

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homodimerize and this association is enhanced through a conformational change in response to

14

the decreased pH in the ampullate gland duct.20,21 This is hypothesized to lead to the formation of

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long multimeric strands21 that then coalesce into fibers through extensive hydrophobic

16

interactions.20

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Spidroin post-translational phosphorylation may also contribute to fiber assembly. NMR

31

P

18

spectrum and amino acid analysis of N. clavipes dragline silk detected phosphotyrosine in the

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dragline silk fiber.22 Eight confirmed tyrosine and serine phosphorylation sites have been

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mapped to MaSp1 sequences that cover the entire repeat domain.23 The possibility of enzyme

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catalyzed phosphorylation/dephosphorylation to control β-sheet assembly of a spidroin-like

22

repeat domain suggests that post-translational phosphorylation may also enhance spidroin

23

solubility by preventing the hydrophobic interaction of the alanine core region.24


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Biomacromolecules

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The use of spider silks as promising biomaterials will require mass production of spider silk-

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like proteins. We previously demonstrated affinity purification of recombinant spider silk-like

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mini-spidroins from transgenic tobacco that could potentially support large scale production.25

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The mini-spidroin monomers produced from tobacco consisted of the native N. clavipes dragline

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silk NTD and CTD flanking 8, 16, or 32 copies of a MaSp1 or MaSp2 consensus block. These

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mini-spidroins can spontaneously form multimers/aggregates of various molecular sizes.

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Spidroins in solution are polyelectrolytes and oppositely charged polyelectrolytes can form

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complexes at their interface (polyelectrolyte complex formation). Although this approach has

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been used to form fibers from poly-L-lysine and gellan gum,26 poly(α,L-glutamic acid) and

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chitosan,27 and lysozyme amyloid nano-fibrils and gellan gum,28 it remains to be determined if it

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can facilitate fiber formation of any spider silk-like proteins.

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Here, we produce mini-spidroin fibers through polyelectrolyte complex formation.

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Recombinant MaSp1 and MaSp2 mini-spidroins containing eight copies of the respective block

14

repeat domains (rMaSp1R8 and rMaSp2R8), after treatment with acetic acid, cross-linking with

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glutaraldehyde, and dilution into phosphate buffer, formed a thin film at the interface with a

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gellan gum solution from which fibers could be immediately pulled with forceps. These

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recombinant spider silk-like fibers were evaluated by single displacement controlled tensile

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testing and several post-spinning treatments were shown to enhance the tensile properties.

19 20


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Biomacromolecules

EXPERIMENTAL SECTION

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Gellan gum and mini-spidroin fiber assembly. The gelatin-like mini-spidroin samples

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resulting from freeze-drying25 were mixed with 25% acetic acid and 25% glutaraldehyde to make

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a spinning solution (dope). For every 25 mg (~25 µl) of gelatin-like liquid (~125 µg/µl and ~190

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µg/µl for rMaSp1R8 and rMaSp2R8, respectively), 0.2 µl of 25% acetic acid and 5 µl of

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glutaraldehyde (25% aqueous solution) was added. The mixture was incubated at ambient

7

temperature for ≥5 hours and then diluted to 150 µl with 10 mM phosphate buffer (pH 7).

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Aliquots of mini-spidroin spinning solution (10 µl to 40 µl, final pH 5.5) were carefully overlaid

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with aqueous gellan gum solution (150 µl to 600 µl) that had been kept at 55°C. The circular

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interface between the two solutions ranged from 3 mm (7.1 mm2) to 4.5 mm (15.9 mm2)

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depending on the volume of the mini-spidroin spinning solution. For rMaSp1 and rMaSp2 alone,

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the gellan gum concentration was 0.5%. For mixtures of rMaSp1 and rMaSp2, the gellan gum

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concentration was either 0.5% or 0.1%. Fibers were drawn from the interface of the gellan gum

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and mini-spidroin mixture, air-dried and kept at room temperature. Bovine serum albumin (BSA,

15

10 mg/ml in water) was used as a negative control.

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Chitosan based mini-spidroin and Gellan gum fiber assembly. Chitosan (medium

17

molecular weight, Sigma Aldrich #448877) was dissolved in 1% acetic acid to a final

18

concentration of 5 mg/ml. To crosslink chitosan, 0.5 ml of glutaraldehyde (25% aqueous

19

solution) was added to 20 ml of the chitosan solution and incubated at room temperature

20

overnight. Mini-spidroin rMaSp1R8 or rMaSp2R8 proteins samples were crosslinked with

21

glutaraldehyde and diluted in phosphate buffer as described above. Fibers with a single mini-

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spidroin component were pulled from the mixture of 20 µl crosslinked chitosan solution and 10

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µl of either rMaSp1R8 or rMaSp2R8 solution, layered with 200 µl 0.5% gellan gum solution.


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Fibers with a combination of rMaSp1R8 and rMaSp2R8 (70:30) were pulled from the mixture of

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chitosan solution (20 µl), rMaSp1R8 (10 µl) and rMaSp2R8 (4.2 µl), layered with 200 µl 0.5%

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gellan gum solution. Fibers were drawn from the interface of the gellan gum and mini-spidroin

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mixture. The fibers were air dried and kept at room temperature.

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Post-spin processing. Air-dried fibers were cut into ~10 cm pieces, immersed in water for 2 minutes, stretched about 15-20 mm while submerged and then air dried again.

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Tensile testing. Fibers were cut into ~3 cm pieces, mounted using cyanoacrylate onto 3 cm2

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paper squares with a precut 1 cm2 opening in the center and equilibrated at room temperature

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(20ºC and 55-65% RH) overnight. The diameter was measured at multiple places along the

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mounted fiber using a Fisher Scientific Micromaster light microscope with a 20x objective and

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an average value used for calculations. The paper square was loaded onto the tensile testing unit

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of a Bruker CETR UMT200 loading cell. The side of the paper square was cut, leaving the fiber

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as the only connection between the upper and lower clamp. A displacement-controlled tensile

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test protocol was used (Pre-load = 0, constant displacement rate = 0.1 mm/s and max

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displacement of 20 mm). The carriage pulling force (Fz) and carriage position (Z) was

16

continuously recorded. Relaxation and thermal drift holding sequence were applied after the max

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displacement was reached. Young’s Modulus and toughness were calculated by UMT-Viewer

18

Software.

19 20


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Biomacromolecules

RESULTS

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Fiber formation conditions with single protein components of rMaSp1R8 or rMaSp2R8 are

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summarized in Table 1. When treated with acetic acid, cross-linked with glutaraldehyde and

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diluted in phosphate buffer, both mini-spidroins resulted in counter-ion condensation at the

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interface with negatively-charged gellan gum and fibrous material was pulled from the thin film

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that was formed (Figure 1). Crosslinking of mini-spidroins by glutaraldehyde is essential for the

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formation of mini-spidroin/gellan gum fibers. Treatment with acid, however, differentially

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affects the ability to pull fibers with rMaSp1 and rMaSp2 proteins. Acid treatment is necessary to

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pull “long” (≥4 inches) fibers with rMaSp1 (without acid only “short” fibers are produced) and

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the opposite is seen with rMaSp2. Light microscope image of these fibers showed that fiber

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surface are rough. Some part of the fibers showed scale-like structures on surface (Figure 2A).

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As has been documented in natural dragline fibers29, air-dried fibers can be rehydrated with

13

water and retain a fiber structure. Neither water nor BSA control reactions exhibited counter-ion

14

condensation; thus, no fibers were assembled.

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16 17

Figure 1. Schematic drawing of the fiber pulling process.

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Biomacromolecules

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Table 1. Fiber assembly of single protein component (rMaSp1R8 or rMaSp2R8) in the presence

2

or absence of acetic acid and/or glutaraldehydea.

3 Acetic acid (25%)

Glutaraldehyde (25% water solution)

Phosphate buffer (pH 7)

Fiber length (every 10 µl spinning solution)

dH2Ob

+

+

+

No Fiber

BSAb

+

+

+

No Fiber

rMaSp1R8

+

+

+

> 4 inches

rMaSp1R8

-

+

+

< 3 inches

rMaSp1R8

+

-

+

No Fiber

rMaSp2R8

+

+

+

< 3 inches

rMaSp2R8

-

+

+

> 4 inches

rMaSp2R8

+

-

+

No Fiber

a

4 5 6 7

Composition of spinning solution is described in Experimental Section. All spinning solution was layered with 0.5% gellan gum solution. b Water and bovine serum albumin (BSA, 10 mg/ml) were used as negative controls.

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Fibers were also pulled from mixtures of rMaSp1 and rMaSp2 proteins. Since rMaSp1R8 only

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formed long fibers when crosslinked in the presence of acetic acid, all rMaSp1R8 samples used

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for composite fibers were treated with acetic acid. For composite fiber formation, rMaSp1R8 and

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rMaSp2R8 were crosslinked with glutaraldehyde and diluted in phosphate buffer (pH 7)

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independently, as described above for single protein fiber formation after which they were mixed

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as indicated in Table 2.

14 15


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Biomacromolecules

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Table 2. Assembly of two protein component fibers (mixture of rMaSp1R8 and rMaSp2R8)

2

using different gellan gum concentrationsa.

3 rMaSp1R8 treated with acetic acid and glutaraldehyde (%)

rMaSp2R8 treated with acetic acid and glutaraldehyde (%)

rMaSp2R8 treated with glutaraldehyde (%)

Phosphate Buffer (pH 7)

Gellan gum (%)

Fiber length (Based on every 10 µl spinning solution)

70

30

-

+

0.5

> 4 inches

70

-

30

+

0.5

> 4 inches

70

30

-

+

0.1

< 2 inches

70

-

30

+

0.1

< 2 inches

4 5

a

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Because the charge density ratio of gellan gum to protein affects the physical property of the

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fiber complexes,28 protein spinning solution was overlaid with two different gellan gum

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concentrations (0.5% or 0.1%). Regardless of whether rMaSp2R8 was crosslinked in the

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presence of acetic acid or not, long composite fibers were formed from the mixture of the two

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proteins at the interface with 0.5% gellan gum but not 0.1% gellan gum (Table 2). Using 1%

11

gellan gum solutions, rMaSp1R8, rMaSp2R8, and mixtures of rMaSp1R8 and rMaSp2R8 mini-

12

spidroins resulted in large globules at the interface that could only be pulled into short thick

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fibers and only short fibers were formed at the lower gellan gum concentration (0.1%). Based on

14

these observations, all future experiments were performed with acetic acid treatment of both

15

rMaSp1 and rMaSp2, glutaraldehyde crosslinking and 0.5% gellan gum.

Composition of spinning solution is described in Experimental Section.

16

When pulling fibers under these conditions, we observed that droplets started to develop along

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the fibers as soon as they were pulled into the air (Figure 2B). A similar phenomenon was

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reported when pulling fibers from the interface of chitosan and poly(α,L-glutamic acid)

19

solution.27 The droplets may represent excess water that is carried with or captured in the fiber


9

Biomacromolecules

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complex. The diameter of the dried mini-spidroin/gellan gum fibers ranged from 20-80 µm

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between droplets and up to 50 to 100 µm at the droplet locations.

3

A

B

4 5 6

Figure 2. Images of mini-spidroin and gellan gum fiber. A. Light microscope images of mini-

7

spidroin and gellan gum fibers taken at 20x with Nikon inverted light microscope. Left,

8

rMaSp1R8 fiber; Middle, rMaSp2R8 fiber; Right, rMaSp1R8 (70%) and rMaSp2R8 (30%)

9

composite fiber. B. Nascent composite fibers from the mixture of rMaSp1R8 (70%) and

10

rMaSp2R8 (30%). Droplets formed along the wet nascent fiber (left) and appear as swollen

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nodes along the dry nascent fiber (right). Mini-spidroin fibers are auto-fluorescent across

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numerous excitation/ emission wavelengths: 408 nm/515 nm, 488 nm/590 nm, and 561 nm/650

13

nm, resembling the autofluorescent property in the range of 400 nm to 630 nm of native Nephila

14

clavipes dragline silks.30 The fibers also showed positive birefringence under polarized light

15

microscopy, another optical property of native dragline silk that denotes its ordered structure.31

16 17

A displacement-controlled tensile test using a Bruker CETR UMT200 microtensile tester was

18

performed and the tensile properties are summarized in Table 3. Fibers produced with rMaSp1R8

19

alone have higher strength and are stiffer (Young’s Modulus) than rMaSp2R8-alone fibers. In


10

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Biomacromolecules

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fact, fibers produced with rMaSp2R8 protein alone were very brittle and, of the 15 samples

2

evaluated, all but three were broken during sample handling prior to tensile testing. Composite

3

fibers possess tensile properties intermediate to the two rMaSp proteins alone (Table 3).

4 5

Table 3. Tensile properties of rMaSpR8 and gellan gum fibers.

6 Fiber Composition

Sample number (N)

Diameter (µm)

Young's Modulus (GPa)

Maximum stress σmax (MPa)

Strain εmax (%)

Toughness (MJ m-3)

rMaSp1R8

10

36.8 ± 9

0.9 ± 0.3

37.9 ± 11.5

7.1 ± 3.5

1.4 ± 1.0

rMaSp2R8

3

54 ± 3

0.2 ± 0.1

12.2 ± 2.4

9.9 ± 5.0

0.6 ± 0.2

rMaSp1/2R8a

13

72 ± 19

0.3 ± 0.2

15.1 ± 5.0

6.8 ± 3.2

0.6 ± 0.4

a

7 8 9

rMaSp1R8 and rMaSp2R8 are crosslinked individually and then mixed in a volume ratio 70:30.

10

Due to the ability to pull fibers using polyelectrolyte complex formation, we considered

11

whether any polymeric materials native to the spider duct might be involved in self-assembly.

12

Davies et al. (2013) detected chitin in the N. edulis major ampullate gland duct matrix by

13

histological staining and Fourier transform infrared (FTIR) spectroscopy.32 Therefore, we

14

investigated the potential of chitosan, a soluble chitinous polysaccharide, to enhance

15

recombinant mini-spidroin fiber production by mixing crosslinked chitosan with crosslinked

16

mini-spidroin spinning solution prior to layering with gellan gum. Interestingly, we were able to

17

pull meters long fibers and fibers could be continuously made until the supply of spinning

18

solution had been exhausted. Chitosan-containing rMaSp1R8 fibers have very similar properties

19

to non-chitosan-containing fibers but both rMaSp2R8 fibers and composite fibers displayed

20

improved strength and toughness with chitosan present in the spinning solution (compare Table 3

21

and Table 4).


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Table 4. Tensile properties of chitosan-containinga rMaSpR8 and gellan gum fibers.

3 Fiber Composition

Sample numbe r (N)

Diameter (µm)



Strain εmax (%)

Toughness (MJ m-3)

rMaSp1R8

9

44.8 ± 10

1.0 ± 0.4

38.5 ± 8.2

9.1 ± 2.8

1.8 ± 0.7

9

34.9 ± 13

0.5 ± 0.2

30.3 ± 10.9

10.9 ± 3.9

1.9 ± 1.2

8

40.6 ± 12

1.4 ± 0.8

45.6 ± 18.3

5.7 ± 1.4

1.2 ± 0.5

rMaSp2R8 rMaSp1/2R8

b

a

4 5 6 7

Composition of chitosan solution is described in the Experimental Section. rMaSp1R8 and rMaSp2R8 are crosslinked individually and then mixed in a volume ratio 70:30.

8

Post-spin processing of man-made spider silk-like fibers by stretching in water has been

9

previously shown to enhance fiber tensile properties.33 Consistent with those observations, the

10

tensile properties of our chitosan-containing fibers are further enhanced by this simple post-spin

11

processing (compare Table 4 and Table 5). The average diameter of the fibers is decreased and

12

the overall toughness is increased for rMaSp1R8 fibers and composite fibers after stretching in

13

water. This is reflected in the increased maximum breaking stress.

b

14 15

Table 5. Tensile properties of post-spin stretched chitosan-containing rMaSpR8 and gellan gum

16

fibers.

17 18 19 20

Fiber Composition

Sample number (N)

Diameter (µm)



Strain εmax (%)

Toughness (MJ m-3)

rMaSp1R8

9

38.4 ± 10

0.8 ± 0.7

65.3 ± 20.5

9.7 ± 2.8

3.2 ± 1.2

rMaSp2R8

9

41 ± 10

1.4 ± 0.5

66.4 ± 22.6

8.9 ± 1.9

2.9 ± 1.3

rMaSp1/2R a

9

35.4 ± 9

1.5 ± 0.8

61.8 ± 14.1

7.4 ± 2.7

2.3 ± 1.0

a

rMaSp1R8 and rMaSp2R8 are crosslinked individually and then mixed in a volume ratio 70:30.


12

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Biomacromolecules

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Figure 3 is a plot of the stress-strain curves showing the low, medium, and high strength

2

representatives of the as-spun gellan-gum/mini-spidroin fibers, as-spun chitosan/gellan-

3

gum/mini-spidroin fibers and post-spin processed chitosan/gellan-gum/mini-spidroin fibers. By

4

adding chitosan, the strength of the rMaSp2R8 fibers and the rMaSp1/2 composite fibers were

5

both improved, but not so much for the rMaSp1R8 fibers (compare left and middle panels). Post-

6

spun processing in water improved the tensile strength of both rMaSp1 and rMaSp2 fibers

7

(compare middle and right panels). Despite the high variance of tensile properties of rMaSp1/2

8

composite fibers, post-spin processing slightly increased their strength and extensibility but the

9

difference is not as clear as the other two types of fibers.

10

11 12

Figure 3. Representative stress-strain curve of gellan gum/mini-spidroin or chitosan/gellan-

13

gum/mini-spidroin fiber. Left, as-spun gellan gum/mini-spidroin fiber, fibers were air-dried;

14

Middle, as-spun chitosan/gellan gum/mini-spidroin fiber, fibers were air-dried; Right, post-spin

15

processed chitosan/gellan gum/mini-spidroin fiber, fibers were air-dried, rehydrated and


13

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 26

1

stretched in water, then air-dried again. Blue, rMaSp1R8 fiber; Yellow, rMaSp2R8 fiber;

2

Magenta, composite fiber from the mixture of rMaSp1R8 (70%) and rMaSp2R8 (30%).

3 4


14

Page 15 of 26

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1

Biomacromolecules

DISCUSSION

2

We have used tobacco-derived mini-spidroin proteins for the production of fibers. For this

3

study, we chose to focus on recombinant proteins containing eight copies of the central block

4

repeat domain to establish basic conditions for fiber production. These mini-spidroins have fairly

5

high solubility in aqueous solution and moisture remains in the purified protein even after freeze-

6

drying.25 Aqueous mini-spidroin solution thickened to a gelatin-like texture after being mixed

7

with acetic acid. Recombinant spider silk-like proteins obtained in previous studies either

8

spontaneously formed fibers-34 or fibers were produced by pulling in a methanol or ethanol

9

coagulation bath after they were dissolved in organic solvent35. The tobacco-derived mini-

10

spidroins that include both N- and C-terminal domains produced no fibers under similar

11

conditions.

12

Spinning these mini-spidroin proteins has been a challenge due to the high water retaining and

13

native folded mini-spidroins. Therefore, a charge attraction-based aqueous spinning method was

14

attempted (polyelectrolyte complex formation). After crosslinking mini-spidroins with

15

glutaraldehyde and diluting with phosphate buffer, the formation of films at the interface of

16

mini-spidroin and gellan gum solutions was observed, from which fibers could be immediately

17

pulled with a pair of forceps (Figures 1 and 2). All fibers displayed positive birefringence and

18

autofluorescence across a broad range of the visible spectrum (data not shown), both of which

19

optical properties of native dragline silk that attest to its ordered structure.31

20

Charge attraction-based polyelectrolyte complex formation is controlled by the charge

21

distribution of each interacting molecule and the ratio of the total charges of each molecule.27,28

22

The gellan gum concentrations used in our experiments are based on those from experiments

23

with poly-glycine26 and poly nano-glutamic acid.28 Using 0.5% gellan gum, we pulled fibers for


15

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

1

individual mini-spidroin proteins rMaSp1R8 and rMaSp2R8 and for mixtures of the two mini-

2

spidroins (Table 3). At gellan gum concentrations above 0.5%, a large globule formed at the

3

interface and pulling resulted in very short, thick fibers that are not suitable for further fiber

4

handling. Lower gellan gum concentrations (0.1%) in our experiment with the combination of

5

rMaSp1 and rMaSp2 mini-spidroins produced very short fibers (less than 2 inches, Table 2).

6

Because different material properties resulted from different gellan gum concentrations, it may

7

be possible to tailor the resulting mini-spidroin complexes for specific purposes. For example,

8

mini-spidroins produced with higher gellan gum concentration may have the potential to form

9

hydrogels or capsules. Such capsule development has been reported using poly-L-lysine.26

10

Glutaraldehyde exists in aqueous solution as multiple forms (monomer, dimer or polymer) and

11

crosslinking reactions, primarily with the nucleophilic ε-amino of lysine side chain,37 are pH and

12

concentration dependent.38 Lysine residues are found only in the NTDs of the purified rMaSps

13

used here (six and four lysine residues in rMaSp1R8 and rMaSp2R8, respectively). It is absent

14

from both MaSp1 and MaSp2 block repeat domains. Assuming lysine residues are the primary

15

target of glutaraldehyde crosslinking, the resulting nanofibrils are expected to be crosslinked by

16

their NTD. Nanofibrils formation is a critical step before large complex could be assembled28.

17

Our results that no fibers were formed if mini-spidroins were not crosslinked by glutaraldehyde

18

provided addition support for this phenomenon (Table 1).

19

In our experiments, each mini-spidroin crosslinking reaction is carried out independently. To

20

pull composite fibers, rMaSp1 and rMaSp2 were mixed after crosslinking. Surprisingly, we

21

could not pull any fibers when rMaSp1 and rMaSp2 were mixed prior to crosslinking. The

22

crosslinking of two mini-spidroins in one reaction could possibly disrupt the size and charge

23

ratio of the molecules so that assembly failed to initiate.


16

Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

Charge distribution and charge density of mini-spidroin molecule is affected by the protonation

2

state. At acidic pH (5.7) the net positive charge that is carried on an rMaSp1R8 molecule

3

(predicted pI of 9.16) will be stronger than that on an rMaSp2R8 molecule (predicted pI of 5.7).

4

This could be the reason that rMaSp1R8 only forms longer fibers when acidified before

5

glutaraldehyde crosslinking (Table 1). Interestingly, long composite fibers could be pulled

6

regardless of whether rMaSp2R8 was treated with acetic acid or not, as long as rMaSp1R8 was

7

treated with acetic acid (Table 2). Considering that MaSp1 protein is dominant in natural Nephila

8

clavipes dragline silk fiber

9

spider major ampullate gland,39 we treated both mini-spidroins with acetic acid in our further

10

6

and that acidification has been shown to occur along the duct of

experiments.

11

Chitin is an abundant biopolymer with β-(1,4)-N-acetyl-D-glucosamine repeat units that

12

provide support strength to cuticles, cell walls and shells for many organisms. Chitin has also

13

been identified in the distal part of the final limb of the Nephila clavipes major ampullate duct32

14

and is proposed to function as a structural support for the draw down taper. However, other

15

forms of chitin may also be present in the duct. Partially deacetylated chitin, chitosan, is a

16

polycationic material at acidic pH that has been used to produce a variety of functional

17

biomaterials. These include fibers and capsules from poly-L-glutamic acid and chitosan,27

18

nanofibrous scaffolds from electrospinning of recombinant spider silk protein, polycaprolactone

19

and chitosan40 and nanogels from chitosan derivatives, hexanoyl chitosan and succinoyl

20

chitosan.41 Chitosan also forms fiber complexes with gellan gum.42 At the gellan gum

21

concentration and crosslinking conditions used in our experiments, chitosan and gellan gum

22

fibers are short and brittle. However, this led us to explore the potential of chitosan and gellan

23

gum to provide a superior support matrix for mini-spidroin fibers. Inclusion of crosslinked


17

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 26

1

chitosan in the mini-spidroin solution allowed meters long fibers to be pulled from the interface

2

of chitosan/mini-spidroin and gellan gum (Table 4) until the spinning solution was depleted. We

3

assume that fibers could be endlessly pulled if the spinning solution were continuously supplied.

4

Mini-spidroin fibers pulled here had average diameters that ranged from approximately 30 to

5

75 µm. Those pulled in the absence of chitosan or any post-processing displayed tensile far

6

inferior to those of native Nephila clavipes dragline silk (tensile strength and toughness < 3.5%

7

and < 1.5% native values, respectively). This result might be anticipated based on the nature of

8

the recombinant proteins that contain both native NTD and CTD domains but only eight copies

9

of the block repeats (native spidroins have ~100 copies of the block repeat). The addition of

10

chitosan to the mini-spidroins spinning solution enhanced the tensile properties of the fibers,

11

particularly those of the rMaSp2R8 and composite fibers (compare Tables 3 and 4). Post-

12

processing by stretching in water resulted in increased strength and overall toughness (Tables 4,

13

5). Post spinning processing also improved the uniformity of fiber diameter.

14

The difference in tensile strength improvement of rMaSp1R8 fiber and rMaSp2R8 fiber

15

in response to post-spin processing may be due to the amino acid difference in the repeat block

16

of the two proteins. Proline, a ring forming amino acid found exclusively in MaSp2 repetitive

17

domain (deprived in MaSp1 repetitive domain) may create inter-molecular spaces required for

18

hydrogen bonding and may affect the nano-structure of the native dragline silk.43 Savage and

19

Gosline reported that the tensile strength of Araneus dragline silk with higher MaSp2 content

20

drastically changed in wet and dry conditions whereas the tensile strength of Nephila dragline

21

silk with lower MaSp2 content did not changed much in wet and dry conditions.44 The difference

22

in tensile strength changes of rMaSp1R8 fiber and rMaSp2R8 fiber in response to water


18

Page 19 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

processing provide corroborating evidence that proline in rMaSp2R8 may play an important role

2

in fiber nanostructure and hydrogen bonding.

3 4

CONCLUSIONS

5

This study builds on our current knowledge on spider silk-like fiber assembly. Through

6

polyelectrolyte complex formation we produced mini-spidroin fibers from tobacco-derived

7

recombinant spidroin-like proteins. This is the first report that man-made spider silk-like fibers

8

can be made through polyelectrolyte complex formation, a cost effective process that is safe for

9

subsequent medical applications. The autofluorescent nature of these fibers is also promising for

10

in vivo visualizing and tracking. The tensile strength of mini-spidroin fibers can be improved by

11

spinning mini-spidroins in a chitosan matrix and, with post-spin processing, we were able to

12

continuously produce fibers with an average diameter in the range of 28 to 33 µm and overall

13

toughness of 3.2 ± 1.2; 2.9 ± 1.3 and 2.3 ± 1.0 MJ m-3 for rMaSp1R8; rMaSp2R8 and rMaSp1/2

14

R8, respectively. We will continue to investigate the self-assembly processes that lead to

15

stronger and tougher fibers that may outperform other man-made materials.

16 17


19

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

AUTHOR INFORMATION

2

Corresponding Author

3

*E-mail: [email protected]

4

Present Addresses

5

‡

6

10708

7

§

8

Point, NC 27268

Page 20 of 26

Department of Human Genetics, One Mead Way, Sarah Lawrence College, Bronxville, NY

Department of Biology, One University Parkway, Congdon 141, High Point University, High

9 10

Notes

11

The authors declare no competing financial interest.

12 13

ACKNOWLEDGEMENTS

14

The authors would like to thank Dr. Michael Ellison from the Department of Material Science

15

and Dr. John Desjardins from the Department of Bioengineering for their help on fiber tensile

16

testing. We would like to thank Dr. Terri Bruce for her help on microscopy and Dr. Florence

17

Tuelé for her constructive suggestions.

18 19


20

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1

Biomacromolecules

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4 5

TABLE OF CONTENTS GRAPHIC

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Polyelectrolyte Fiber Assembly of Plant-Derived Spider Silk-like Proteins

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