Recombinant Spidroins Fully Replicate Primary Mechanical Properties

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Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk Christopher H. Bowen, Bin Dai, Cameron J. Sargent, Wenqin Bai, Pranay Ladiwala, Huibao Feng, Wenwen Huang, David L Kaplan, Jonathan M. Galazka, and Fuzhong Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00980 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Biomacromolecules

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TITLE: Recombinant Spidroins Fully Replicate Primary Mechanical

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Properties of Natural Spider Silk

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AUTHORS: Christopher H. Bowen1,†, Bin Dai1,†, Cameron J. Sargent2, Wenqin Bai1, Pranay

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Ladiwala1, Huibao Feng1, Wenwen Huang4, David L. Kaplan4, Jonathan M. Galazka5, Fuzhong

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Zhang1-3,*

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AFFILIATIONS:

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1

Department of Energy, Environmental and Chemical Engineering,

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2

Division of Biological & Biomedical Sciences,

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3

Institute of Materials Science & Engineering,

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Washington University in St. Louis, Saint Louis, MO 63130, USA

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Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA

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5

Space Biosciences Division, Ames Research Center, National Aeronautics and Space

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Administration, Moffett Field, CA 94035, USA

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†

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*

These authors contributed equally. Correspondence to: [email protected]

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ABSTRACT:

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Despite significant efforts to engineer their heterologous production, recombinant spider

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silk proteins (spidroins) have yet to replicate the unparalleled combination of high strength and

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toughness exhibited by natural spider silks, preventing their use in numerous mechanically-

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demanding applications. To overcome this long-standing challenge, we have developed a

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synthetic biology approach combining standardized DNA part assembly and split intein-

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mediated ligation to produce recombinant spidroins of previously unobtainable size (556 kDa),

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containing 192 repeat motifs of the Nephila clavipes dragline spidroin. Fibers spun from our

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synthetic spidroins are the first to fully replicate the mechanical performance of their natural

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counterparts by all common metrics, i.e. tensile strength (1.03 ± 0.11 GPa), modulus (13.7 ± 3.0

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GPa), extensibility (18 ± 6%), and toughness (114 ± 51 MJ/m3). The developed process reveals a

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path to more dependable production of high-performance silks for mechanically-demanding

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applications while also providing a platform to facilitate production of other high-performance

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natural materials.

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INTRODUCTION:

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Dragline spider silk is among the strongest and toughest biological materials, capable of

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outperforming most synthetic polymers and even some metal alloys.1-3 These remarkable

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properties have resulted in a growing list of potential high-performance material applications for

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spider silks ranging from impact resistant textiles to monofilament surgical sutures;

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unfortunately, the impracticalities of spider farming have long restricted the practical

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development of such applications.2,4 The resulting demand for a dependable source of high-

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performance silk fibers has driven both academic and industrial research toward production of

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recombinant spidroins in engineered hosts including bacteria, yeasts, plants, and animals.5-9

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While providing significant technical and scientific advances, these efforts have so far been

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unable to yield recombinant silk fibers that fully replicate natural spider silk's unparalleled

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combination of mechanical properties (i.e. high strength, modulus, extensibility, and toughness),

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due in part to an inability to stably produce highly repetitive, high molecular weight (MW)

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proteins in heterologous hosts.2,4,9

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Natural dragline spidroins are typically very large (>300 kDa), highly repetitive proteins

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that contain hundreds of tandem repeats of glycine- and alanine-rich sequences.10,11 As with most

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polymers, the size of these spidroins is expected to positively correlate with tensile strength (up

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to a limiting MW) due to an increased density of interchain interactions and entanglements and

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fewer chain-end defects.9,12 Indeed, previous work has demonstrated a clear correlation between

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MW and strength for recombinant Nephila clavipes dragline fibers, with the largest spidroin (96-

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mer, 285 kDa) yielding the strongest recombinant fiber reported to date (σ ≈ 550 MPa).9

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However, the upper limit for the strength-to-MW relationship in spidroins is currently unclear

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and despite the apparent need for even larger spidroins to yield fibers with mechanical properties

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on par with natural spider silk (σ = 0.8-1.2 GPa for N. clavipes dragline, see Table S1),

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heterologous production of spidroins larger than 285 kDa in quantities sufficient for fiber testing

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has proven to be extremely difficult.9 Challenges of heterologous production arise from a series

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of issues including genetic instability of long and highly repetitive DNA sequences, translation

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inhibition by complex mRNA secondary structures, a high demand for glycine and alanine

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tRNAs, and overall metabolic burden caused by spidroin overexpression.4,9 To overcome these

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long-standing challenges, we envisioned a synthetic biology platform employing standardized

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DNA part assembly to facilitate covalent, post-translational ligation of the largest spidroins that



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can be stably expressed in engineered Escherichia coli (i.e. 96-mer), permitting production of

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spidroins of previously unobtainable MW that can be used to generate fibers with exceptional

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mechanical properties (Figure 1).

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Of the various biochemical approaches available for post-translational protein ligation,

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split inteins (SIs) are perhaps the best suited for generating high MW proteins for material

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applications. SIs are peptide auto-processing domains that, when fused to separately expressed

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proteins, catalyze spontaneous splicing reactions, covalently linking their fusion partners via a

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peptide bond and leaving only a few residues (≤ 6) at the ligation site.13 When SIs are used to

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ligate 96-mer spidroins, these residues are unlikely to affect the properties of the much larger

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ligated product (6720 amino acids total). Other common approaches for post translational

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ligation involve either side chain bonding (e.g. SpyTag-SpyCatcher), which produces branched

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structures that could negatively affect material properties, or catalysis by separately expressed

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proteins (e.g. Sortase A), which increases process complexity.14 For these reasons, we chose to

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employ SI-based ligations for our envisioned platform, such that ligation of an N-intein (IntN)-

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fused 96-mer spidroin (96N) with a C-intein (IntC)-fused 96-mer (C96) would yield a 556 kDa,

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192-mer spidroin (Figure 1e).


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Figure 1. Process schematic for split intein-mediated ligation of spider silk proteins with

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unprecedented molecular weight and mechanical properties. (a) The highly repetitive core of

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natural N. clavipes dragline silk protein MaSp1 (shown as a simplified consensus peptide

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sequence) was reduced to a single repeat unit (1-mer). Note, while "-mer" often refers to a small

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molecule or individual amino acid, the abbreviation here refers to the 35 amino acid peptide that

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constitutes the repeating unit of the larger spidroin. (b) The 1-mer DNA sequence was combined

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in silico with 5' UTR, RBS, split intein (SI), and terminator sequences, which were then

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computationally optimized for microbial production. (c) The optimized DNA sequences were

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assembled within the framework of a standardized DNA part assembly system termed SI-Bricks

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to yield complementary IntC- or IntN-fused 96-mer constructs. (d) Constructs were transformed

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to metabolically engineered E. coli for bioproduction. (e) Cell cultures were mixed and lysed to

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initiate SI-mediated covalent ligation of 96-mer spidroins to yield a 192-mer, 556 kDa product.

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(f) The ligated product was purified and spun into fibers for mechanical testing. Scale bar

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indicates 5 µm.

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MATERIALS AND METHODS:

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Strains and growth conditions. E. coli NEB 10-beta (NEB10β) was used for all plasmid

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cloning and protein production. For all cloning, E. coli strains were cultured in Terrific Broth

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(TB) containing 24 g/L yeast extract, 20 g/L tryptone, 0.4% v/v glycerol, 17 mM KH2PO4, and

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72 mM K2HPO4 at 37 °C with appropriate antibiotics (50 µg/mL kanamycin and 30 µg/mL

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chloramphenicol). M9 glucose medium with tryptone supplement (2% w/v glucose, 1x M9 Salts,

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75 mM MOPS pH 7.4, 12 g/L tryptone, 5 mM sodium citrate, 2 mM MgSO4·7H2O, 100 µM

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FeSO4·7H2O, 100 µM CaCl2·2H2O, 3 µM thiamine, 1x micronutrients [40 µM ZnSO4·7H2O, 20

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µM CuSO4·5H2O, 10 µM MnCl2·4H2O, 4 µM H3BO3, 0.4 µM (NH4)6Mo7O24·4H2O, and 0.3 µM

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CoCl2·6H2O]) was used for protein production in bioreactors.

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Chemicals and reagents. Unless otherwise noted, all chemicals and reagents were

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obtained from MilliporeSigma. Plasmid purification and gel extraction kits were purchased from

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iNtRON Biotechnology. FastDigest restriction enzymes and T4 DNA ligase were purchased

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from Thermo Fisher Scientific and used for all digestions and ligations following the

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manufacturer's suggested protocols.

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Genetic assembly of 64- and 96-mer spidroins. The multimeric spidroin DNA

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sequences were constructed as SI-Bricks genetic parts based on a method modified from

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previous work (Supplementary Note 1, Figure S1, Figure S2).5,9 Specifically, an optimized

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coding sequence of 1-mer N. clavipes MaSp1 was chemically synthesized by Integrated DNA

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Technologies. To fit the SI-Bricks standard, the DNA sequence was flanked on the 5' end by

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restriction sites 5'-KpnI/NheI-3' and on the 3' end by restriction sites 5'-SpeI/Kpn2I-3'. The

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sequence was inserted between restriction sites KpnI/Kpn2I of a medium copy (pBBR1

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replication origin), chloramphenicol resistance (CmR) SI-Bricks expression vector, resulting in


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plasmid p1 (Figure S2b). To begin the recursive directional genetic assembly, plasmid p1 was

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linearized by digestion with NheI and ligated with another 1-mer sequence digested by

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NheI/SpeI. The ligation was transformed to NEB10β for amplification, yielding plasmid p2

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containing 2-mer spidroin (Figure S2c). The same procedure was repeated for p2, with insertion

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of a linearized 2-mer sequence, to yield plasmid p4, and the process was repeated until p64 (32-

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mer + 32-mer) and p96 (64-mer + 32-mer) were obtained (Figure S2d). Because the annealing of

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NheI/SpeI overhangs produces a non-cutting sequence, digestion with NheI was used to confirm

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proper insert direction after each ligation iteration.

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Construction and sequence optimization of silk-SI-fusion proteins. N- and C-intein

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amino acid sequences (CfaN and CfaC, respectively) were obtained from a recent publication15

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and prepared as SI-Bricks standard parts as described below. To ensure substantial production of

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the final SI-fused silk proteins, the SI coding sequences were optimized for E. coli expression

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within the genetic context of all flanking sequences using a combination of computational

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approaches.16,17 Specifically, an initial DNA sequence was computationally designed that

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contains (from 5’ to 3’) a 5' UTR/RBS containing the EcoRI/BglII sites, a short coding sequence

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(5'-ATGGCTAAGACTAAA-3') intended to increase translation initiation rate as described

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previously,18 the CfaC coding sequence, the KpnI/NheI sites, and the multimeric silk sequence.

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Within this genetic context, only the CfaC sequence was set variable and optimized using a

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modified E. coli codon usage table with extra weight given to 5' mRNA structure minimization

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within the sequence optimization algorithm.19 The resulting optimized CfaC sequence (including

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5' UTR/RBS and necessary SI-Bricks restriction sites; Table S2) was synthesized as a gblock

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fragment by Integrated DNA Technologies and was inserted 5' of the 64-mer or 96-mer

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sequences by digestion/ligation with EcoRI/KpnI to yield plasmids pC64 and pC96, which encode



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fusion proteins C64 and C96, respectively (Table S3, Figure S2e). Similarly, to codon-optimize

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CfaN, an initial DNA sequence was computationally designed containing (from 5’ to 3’) the

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multimeric silk sequence, the SpeI/Kpn2I restriction sites, a CfaN coding sequence, and the

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BamHI/XhoI sites. Within this context, only the CfaN coding sequence was subjected to the

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optimization process. The resulting CfaN sequence (Table S2), including SI-Bricks restriction

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sites, was synthesized and inserted 3' of the 64-mer and 96-mer sequences by digestion/ligation

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with Kpn2I/XhoI to yield plasmids p64N and p96N, which encode fusion proteins 64N and 96N,

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respectively (Figure S2e). The resulting SI-Bricks parts/vectors allow for easy swapping of any

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IntC, multimeric material protein, or IntN of interest by digestion with EcoRI/KpnI, KpnI/Kpn2I,

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or Kpn2I/XhoI, respectively. Additionally, the presence of NheI/SpeI sites allows for in situ

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recursive directional genetic assembly of any large, repetitive material proteins of interest

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(Supplementary Note 1, Figure S1, Figure S2). Upregulation of GlyV tRNA production. In addition to sequence optimizations, cellular

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glycyl

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spidroin overexpression. The glyV tRNA coding sequence and its native promoter were PCR-

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amplified from the NEB10β genomic DNA (Table S2) and cloned between AatII/XhoI sites of a

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medium copy vector carrying p15A replication origin and Kanamycin resistance (KanR), yielding

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plasmid pGlyV (Table S3). For all spidroin expression, pGlyV was co-transformed with the

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spidroin plasmid.

tRNA levels were also upregulated to meet the high demands on

glycyl

tRNA posed by

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Shake flask cultures. For initial ligation tests (Figure S3), protein production was carried

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out in shake flasks. Transformants were cultured overnight in 50 mL TB medium at 37 °C on an

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orbital shaker. Overnight 50 mL cultures were then used to inoculate 500 mL fresh TB medium

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in Erlenmeyer flasks at an initial OD600 = 0.08. Cultures were grown at 37 °C with orbital


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shaking to OD600 = 6, then induced by addition of 1 mM IPTG and cultured for an additional 6

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hours at 30 °C with orbital shaking.

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Bioproduction in fed-batch bioreactors. All spidroins were ultimately produced in 2 L

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fed-batch bioreactors (Bioflo120, Eppendorf). Transformants were cultured overnight in 50 mL

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TB medium at 37 °C on an orbital shaker. The overnight cultures were then used to inoculate 1 L

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glucose M9 medium with tryptone supplement at an initial OD600 = 0.08. After overnight

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incubation at 37 °C with orbital shaking, 1 L cultures were pelleted by centrifugation at 4500 x g

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for 10 min and resuspended in 300 mL sterile resuspension medium (250 mM MOPS pH 7.4,

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2.5% w/v glucose, 60 g/L tryptone, 25 mM sodium citrate, 10 mM MgSO4, 500 µM

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FeSO4·7H2O, 15 µM thiamine, 5x micronutrients). The resuspended cultures were then added to

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an autoclaved 2 L Bioflo120 heat-blanketed bioreactor containing 1.2 L water and 1.15x M9

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salts. Sterile CaCl2·2H2O was added to a final concentration of 100 µM. Antifoam 204 was

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added as needed to minimize foaming (approximately 0.01% v/v). Agitation and air flow was

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regulated to maintain approximately 70% dissolved oxygen (DO). After consumption of the

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initial 0.5% w/v glucose (as judged by ∆DO), a sterile substrate feed (20% w/v glucose, 48 g/L

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tryptone, and 10 g/L MgSO4·7H2O) was initiated to maintain a linear growth rate. Reactors were

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induced at OD600 = 80 by addition of 1 mM IPTG and culture temperature was reduced to 30 °C.

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Cultures were collected four hours after induction. Based on densitometric analysis of

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Coomassie Blue-stained SDS-PAGE gels (Figure S4), final product titer for 96-mer reactants

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was estimated at approximately 2 g/L or 63 mg/g cell dry weight.

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Protein ligation. Cell cultures were pelleted by centrifugation at 4500 x g for 30 min.

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Pellets from complimentary SI-fused spidroins (e.g. 96N and C96 or 64N and C64) were combined

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at a 1:1 reactant ratio based on densitometric analysis of Coomassie Blue-stained SDS-PAGE



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gels. Mixed pellets were resuspended in sonication buffer (300 mM NaCl, 20 mM MOPS pH

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7.4, 2 mM TCEP, 1 mM PMSF) and sonicated using a QSonica Q700 sonicator (Qsonica) for 10

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min (5s on, 10s off). Sonicated resuspensions were pelleted by centrifugation at 25,000 x g for

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30 min to remove supernatants. Pellets were resuspended in ligation buffer (8 M urea, 20 mM

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MOPS pH 7.4, 300 mM NaCl, and 2 mM TCEP) and stirred at 37 °C for 24 h to dissolve SI-

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fused spidroins and ensure complete ligation. The mixtures were then centrifuged at 25,000 x g

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for 1 h to remove cell debris and undissolved proteins.

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Protein purification. The purification protocol was modified from previous methods.9

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Specifically, ligated spidroins in ligation buffer were acidified to pH 4.0 with acetic acid.

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Ammonium sulfate was then added to a final concentration of 1.2 M. The mixture was then

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centrifuged at 40,000 x g for 30 min. The pellet was discarded, and additional ammonium sulfate

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was added to the supernatant to a final concentration of 2.3 M. After stirring for 1 h, the mixture

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was centrifuged again at 40,000 x g for 15 min., yielding a pellet containing approximately 75%

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spidroin as estimated by Coomassie Blue-stained SDS-PAGE gels. The supernatant was

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discarded, and the pellet was resuspended in SEC buffer (8 M urea, 10 mM ammonium

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bicarbonate pH 10) for further purification by size-exclusion chromatography. SEC purifications

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were performed on an AKTA Pure Chromatography System (GE Healthcare Life Sciences)

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using a HiPrep 16/60 Sephacryl S-500 HR column (for 128-mer and 192-mer) or a HiPrep 16/60

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Sephacryl S-400 HR column (for 96-mer). Proteins were separated using an isocratic elution

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with SEC buffer at a flow rate of 0.5 mL/min. Fractions containing greater than 90% ligation

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product, as determined by SDS-PAGE gel densitometry, were collected, resulting in

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approximately 5% final product recovery as judged by product dry mass. SEC-purified fractions


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were combined and dialyzed in 10K MWCO SnakeSkin dialysis tubing (ThermoFisher

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Scientific), followed by lyophilization.

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Ligation kinetics analysis. For kinetics analysis, 64-mer protein concentrations in crude

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lysates were estimated by densitometric analysis of Coomassie Blue-stained SDS-PAGE gels.

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Based on estimated concentrations, fully sonicated resuspensions of 64C and N64 in ligation

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buffer were combined to give final concentrations of 100 µM for both 64C and N64 in a final

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volume of 500 µL. These mixtures were pelleted by centrifugation, and pellets were resuspended

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in 500 µL of desired test buffer pre-incubated at the desired test temperature. Reactions were

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quenched by transferring 5 µL of reaction to 95 µL of Laemmli sample buffer preheated to 100

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°C and continuing boiling for 10 min.

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SDS-PAGE and densitometric analysis. All SDS-PAGE gels were 1 mm thick,

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discontinuous with 3% stacking gel, and hand cast at the indicated percentages. Samples were

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prepared at 1 mg/mL or 5 µM total protein in Laemmli sample buffer (2% SDS, 10% glycerol,

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60 mM Tris pH 6.8, 0.01% bromophenol blue, 100 µM DTT). Gels were run on Mini-

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PROTEAN Tetra Cells (Bio-Rad) in 1x Tris-glycine SDS buffer (25 mM Tris base, 250 mM

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glycine, 0.1% w/v SDS), until just before the dye front exited the gel. Gels were stained in

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Coomassie Blue solution (50% methanol, 10% w/v acetic acid, 1 g/L Coomassie Brilliant Blue)

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for a minimum of one hour at room temperature with gentle agitation and destained in

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Coomassie Blue destain buffer (40% v/v methanol, 10% v/v acetic acid) for a minimum of one

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hour. Gels were imaged on an Azure c600 Imager (Azure Biosystems). All densitometry analysis

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was performed with the AzureSpot Analysis Software (Azure Biosystems). Images were

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background subtracted with a built-in automatic lane edge subtraction algorithm. Protein band

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intensities were integrated by the AzureSpot software. Ligation yield was calculated as the



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integrated intensity of the product band over the sum of both reactant and product band areas.

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Purity was calculated as the integrated intensity of the product band over the integrated intensity

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of all other bands. Spidroin titer was calculated as the integrated intensity of the spidroin band

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over the integrated intensity of all other bands multiplied by the measured cell density (OD600) of

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culture at the time of sampling and 150 mg/L/OD600 (150 mg/L/OD600 is an average typical total

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protein titer for E. coli grown in glucose minimal medium).20

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Fiber spinning and mechanical testing. Fiber spinning and mechanical testing were

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performed following a protocol modified from previous methods.9,21,22 Lyophilized spidroin

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powders were dissolved in hexafluorisopropanol (HFIP) to 17% w/v. This protein dope was

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loaded to a 100 µL Hamilton gastight syringe (Hamilton Robotics) fitted with a 23s gauge (116

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µm inner diameter, 1.71 inch length) needle. The syringe was fitted to a Harvard Apparatus

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Pump 11 Elite syringe pump (Harvard Apparatus), and the dope was extruded into a 95% v/v

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methanol bath at 5 µL/min. Extruded fibers were then transferred to a 75% v/v methanol bath

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and carefully extended at approximately 1 cm/s to the maximum draw ratio (~600%) without

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fiber fracture. Extended fibers were removed from the bath and held under tension until visibly

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dry. Segments of post-drawn fibers (20 mm) were carefully laid exactly vertical across a 5 mm

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(vertical) x 15 mm (horizontal) opening cut into a 20 mm x 20 mm piece of cardstock and fixed

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with adhesive tape at both ends of the opening. Diameters of mounted fibers were then measured

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by light microscopy, averaging measurements at three points along the fiber axis (Tables S4-S6,

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Figure S5a). Mechanical properties were measured by axial pull tests on an MTS Criterion

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Model 41 universal test frame fitted with a 1 N load cell (MTS Systems Corporation). Cardstock

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holders were mounted between two opposing spring-loaded grips, and the supporting edges were

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carefully cut. Pull tests were conducted at a relative humidity of 30% and temperature of 22 °C,


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Biomacromolecules

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with a constant crosshead speed of 10 mm/min. Stress-strain curves were recorded by the MTS

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TW Elite test suite at a sampling rate of 50 Hz. Fiber breaks were recorded when a 90% drop

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from peak stress was detected. All mechanical properties were automatically calculated by the

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MTS TW Elite test suite. Ultimate tensile strength was calculated as the maximum measured

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load over the initial fiber cross-sectional area (‫ ݎߨ = ܣ‬ଶ ), as determined from measured initial

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diameters (see Supplementary Note 2 for an analysis of the validity of this approach). Modulus

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was calculated as the slope of a linear least squares fit to the stress/strain data of the initial elastic

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region. Toughness was calculated as the area under the total stress/strain curve divided by the

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initial fiber volume (ܸ = ߨ‫ ݎ‬ଶ ℎ) as calculated from measured initial fiber diameters and set initial

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gage length of 5 mm. For each protein, a total of 14 fibers were measured in this manner.

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Light microscopy. Fiber diameters were measured using images acquired with a Zeiss

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Axio Observer ZI Inverted Microscope equipped with a phase contrast 20x objective lens and the

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Axiovision LE software (Zeiss). For morphological analysis and further confirmation of fiber

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diameters, additional images were acquired with a Nikon Eclipse TiE Inverted Microscope

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equipped with a 60x objective and analyzed using the Nis-Elements software (Nikon; Figure

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S5a).

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Scanning Electron Microscopy. Following tensile tests, silk fibers were mounted onto a

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sample holder using conductive tape. The sample holder was sputter coated with a 10 nm gold

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layer using a Leica EM ACE600 high vacuum sputter coater (Leica Microsystems). Fibers were

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imaged using a Nova NanoSEM 230 Field Emission Scanning Electron Microscope (Field

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Electron and Ion Company, FEI) at an accelerating voltage of 7-10 kV. Fiber surface roughness

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(both exterior and interior) was calculated using MountainsMap SEM Topo software (Digital

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Surf). The root mean square height (Sq) of each surface was determined from single images



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using greyscale values, normalized by the entire range of values in each micrograph. To remove

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fiber curvature from consideration, a standard Gaussian filter with a threshold of 2.5 µm was

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applied when analyzing exterior surfaces. For convenience, exterior and interior surface

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roughness values are presented relative to each other, with the highest interior value set at 1.

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Fiber circularity was calculated from cross-sectional areas and perimeters as 4ߨ(௣௘௥௜௠௘௧௘௥ మ ),

290

where a perfect circle gives a value of 1.

௔௥௘௔

291

Polarized Raman spectromicroscopy. The method reported here is adapted from

292

several previous studies of molecular alignment in spider silk fibers.23,24,25 Silk fibers were

293

carefully fixed to glass microscope slides with microscale markings to ensure that spectra were

294

acquired at the same location before and after stage rotation. Raman spectra were acquired with a

295

Renishaw RM1000 InVia Confocal Raman Spectrometer (Renishaw) coupled to a Leica DM LM

296

microscope with rotating stage (Leica Microsystems). Silk fibers were initially oriented along the

297

x-axis. Fibers were irradiated at a fixed point with the 514 nm line of an argon laser with

298

polarization fixed along the x-axis and focused through a 50x objective (NA = 0.75). Spectra

299

were recorded from 1150-1750 cm-1 with an 1800 lines/mm grating. For each acquisition, a total

300

of 16 spectra were accumulated, each for 10 s. The stage was then rotated to orient fibers along

301

the y-axis with the same laser polarization, and spectra were acquired a second time at the same

302

fixed point. No signs of thermal degradation were apparent either visually or within recorded

303

spectra. All recorded spectra were analyzed using Fityk 0.9.8.26 Baselines were subtracted from

304

all spectra using the built-in Fityk automatic convex hull algorithm. For intensity ratio

305

calculations, all spectra were normalized to the intensity of the 1450 cm-1 peak, which arises

306

from CH2 bending and is insensitive to protein conformation.24 For each fiber, the normalized

307

intensity of the peak at 1670 cm-1 when oriented along the Y-axis was divided by the normalized


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Biomacromolecules

௒

308

intensity of the peak when oriented along the X-axis to give the intensity ratio (‫ ܫ‬௑). This

309

procedure was performed on a total of three separate fibers and calculated intensity ratios were

310

averaged. Spectra were also averaged and presented in Figure 3d with standard deviations for

311

each point along the spectra.

312

Fourier Transform Infrared Spectroscopy. For secondary structure determination,

313

FTIR spectra were acquired with a Thermo Nicolet 470 FT-IR spectrometer (ThermoFisher

314

Scientific) fitted with a Smart Performer ATR accessory with Ge crystal. Spectra were acquired

315

from 1350-1750 cm-1 at 4 cm-1 resolution. A total of 254 scans were accumulated for each

316

sample. All recorded spectra were analyzed using Fityk 0.9.8. Baselines were subtracted from all

317

spectra using the built-in Fityk convex hull algorithm. The amide I band (1600 - 1700 cm-1) was

318

deconvolved into a set of five Lorentzian peaks centered at 1626.5, 1646.5, 1659, 1679.5, and

319

1700 cm-1 for β-sheet, random coil, α-helix, β-turn, and β-sheet components, respectively.

320

Component peak assignments were based on those used in previous studies.27 Peak areas were

321

integrated and percentages were calculated as the component peak area over the sum of all peak

322

areas. Percentages were averaged from measurements of three fibers.

323

Statistical analysis. GraphPad Prism 7 (GraphPad Software) was used for statistical data

324

analysis, using both two-tailed unpaired t-tests (26 degrees of freedom) and one-way ANOVA

325

tests (41 degrees of freedom) to compare data sets.

326 327

RESULTS AND DISCUSSION:

328

To facilitate microbial production of highly repetitive, SI-fused material proteins, we

329

developed a standardized DNA part assembly system termed SI-Bricks (Supplementary Note 1,

330

Figure S1). SI-Bricks allows for rapid genetic swapping of the core components of the



331

envisioned SI-mediated ligation platform (i.e. N-inteins, material proteins, C-inteins, and fusion

332

domains/purification tags) in addition to common standardized biological parts (e.g. promoters,

333

ribosomal binding sites, replication origins, and selection markers) (Table S7), all by means of

334

restriction enzyme digestion and enzymatic ligation. To construct the SI-Bricks parts necessary

335

for this study, we first assembled 64-mer and 96-mer spidroin DNA sequences by recursive

336

directional genetic assembly of a single, codon-optimized repeat unit (1-mer) of the N. clavipes

337

dragline spidroin MaSp1.5 The assembled spidroin sequences, flanked by SI-Bricks restriction

338

sites, were genetically combined with codon-optimized SI DNA sequences and other necessary

339

expression parts (Figure S2). Given the tendency of high MW spidroins to form inclusion bodies

340

in microbial hosts, we employed a recently engineered SI pair (Cfa) that retains catalytic activity

341

in the presence of 8 M urea,15 a denaturant often used to extract and solubilize spidroins from E.

342

coli. The resulting SI-fused spidroins (64N, C64, 96N, and C96) were individually expressed in an

343

E. coli host with

344

glycine codons in the spidroin sequences. Using our optimized fermentation conditions, typical

345

titers of the SI-fused spidroins were approximately 2 g/L from a glucose minimal medium with

346

tryptone supplementation after four hours of production (Figure S4).

glycyl

tRNA levels engineered to meet the demands of the most frequently used

347

Spidroin inter- and intramolecular interactions can be highly sensitive to salt and pH,

348

even in the presence of 8 M urea28,29 and we expected that such interactions would lower ligation

349

efficiency of the Cfa SIs. Thus, to test optimum conditions for the ligation of Cfa-fused

350

spidroins, 8 M urea extracts of E. coli expressing 64N or C64 were mixed at several salt

351

concentrations, temperatures, and pH values (Figure S3). Under all tested conditions, SI-

352

mediated spidroin ligation was both rapid and robust, with the highest ligation yields observed at

353

37 °C, 300 mM NaCl, pH 7. Thus, for all subsequent ligations, these conditions were maintained,


Page 16 of 29

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Biomacromolecules

354

giving ligation yields of 68% and 62% for 128-mer and 192-mer spidroins, respectively (Figure

355

2a,b).

356

Ligation products were initially separated from most cellular proteins by selective

357

ammonium sulfate precipitation and then further separated from unreacted 64-mer or 96-mer by

358

size exclusion chromatography (SEC) for a final product purity ≥90% (Figure S6). As a standard

359

for mechanical properties, a 96-mer spidroin with no SIs was also expressed and purified

360

following identical procedures. Spinning dope was prepared from lyophilized powder of each

361

purified protein and fibers were spun and mechanically tested following well-documented wet-

362

spinning protocols detailed in the Materials and Methods section.

363

Comparing post-drawn 96-mer and 192-mer fibers, mechanical testing revealed

364

significant, nearly two-fold increases in both tensile strength (from 525 to 1031 MPa, P

Recombinant Spidroins Fully Replicate Primary Mechanical Properties

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