Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin

Abstract

Spider dragline silk is a remarkable material that shows excellent mechanical properties, diverse applications, biocompatibility and biodegradability. Transgenic silkworm technology was used to obtain four types of chimeric silkworm/spider (termed composite) silk fibres, including different lengths of recombinant Major ampullate Spidroin1 (re-MaSp1) or recombinant Major ampullate Spidroin2 (re-MaSp2) from the black widow spider, Latrodectus hesperus. The results showed that the overall mechanical properties of composite silk fibres improved as the re-MaSp1 chain length increased, and there were significant linear relationships between the mechanical properties and the re-MaSp1 chain length (p < 0.01). Additionally, a stronger tensile strength was observed for the composite silk fibres that included re-MaSp1, which only contained one type of repetitive motif, (GA)_n/A_n, to provide tensile strength, compared with the silk fibres that includedre-MaSp2, which has the same protein chain length as re-MaSp1 but contains multiple types of repetitive motifs, GPGXX and (GA)_n/A_n. Therefore, the results indicated that the nature of various repetitive motifs in the primary structure played an important role in imparting excellent mechanical properties to the protein-based silk fibres. A silk protein with a single type of repetitive motif and sufficiently long chains was determined to be an additional indispensable factor. Thus, this study forms a foundation for designing and optimizing the structure of re-silk protein using a heterologous expression system.

Figure 1

Schematic representation of expression cassettes in the three piggyBac-derived transgenic vectors harbouring re-MaSp1 sequences of different lengths. A schematic of pBac[3 × P3-DsRed]-MaSp1 × 2 (a), pBac[3 × P3-DsRed]-MaSp1 × 12 (b) and pBac[3 × P3-DsRed]-MaSp1 × 16 (c) vectors used to express re-MaSp1, including 2-,12- and 16-fold typical repetitive units of MaSp1, respectively. (d) The key elements are shown using boxes with different colours. Fib-H promoter: the primary promoter of Fib-H (1269 bp); SP: the signal peptide of Fib-H; CTD of MaSp1: C-terminal domain of the MaSp1; Fib-HCTD and polyA: the partial C-terminal domain and polyA signal of the Fib-H; 3 × P3-DsRed was used as the marker gene for screening positive individuals.

Figure 2

Positive transgenic strains for DsRed-specific expression in the eyes. (a,a’) G1 egg, (b,b’) larvae and (c,c’) moth of the WT strains were viewed under white light and red fluorescence excitation wavelengths, respectively. (d,d’)G1 egg, (e,e’) larvae and (f,f’) moth of the DsRed-positive strains were viewed under white light and red fluorescence excitation wavelengths.

Figure 3

Schematic diagram for generating heterozygous and homozygous transgenic silkworm lineages.

Figure 4

Expression analysis of different lengths of exogenous re-MaSp1. (a) The relative expression analysis of different lengths ofre-MaSp1in the PSGs of the 3^rd day of the fifth instar larvae in G4 heterozygous transgenic lineages was performed by qRT-PCR. Mean ± SD values were derived from three independent replicate experiments. Gradient sodium dodecyl sulfate-polyacrylamide gel (5%-12%SDS-PAGE) analysis of the degummed silkworm cocoons was performed, followed by immunoblotting on nitrocellulose membranes; M: 250 kDa protein Marker; CK: the degummed silkworm cocoons of wild-type Lan10; (b,c) the degummed silkworm cocoons of MASP1-2-1 and MASP1-2-2 transgenic lineages; (b’,c’) the degummed silkworm cocoons of MASP1-12-1, MASP1-12-5, MASP1-12-8 and MASP1-12-10 transgenic lineages; (b”,c”) the degummed silkworm cocoons of MASP1-16-2, MASP1-16-6, MASP1-16-8 and MASP1-16-14 transgenic lineages. (d) Expression analysis of the expression of different lengths of re-MaSp1 relative to the Fib-L protein in homozygous G5 through grey analysis using the Gel-Pro-analyzer4 software. Mean ± SD was derived from three independent replicate experiments.

Figure 5

Field emission scanning electron micrographs of the fibres. The surface structure of the silk fibres derived from the non-transgenic silkworm lineages (a) and the transgenic silkworm lineages MASP1-2-1 (b), MASP1-12-5 (c) and MASP1-16-2 (d); the cross section structure of the silk fibres derived from the non-transgenic silkworm lineages (a’,a”) and the transgenic silkworm lineages MASP1-2-1(b’,b”), MASP1-12-5 (c’,c”) and MASP1-16-2 (d’,d”). Scale bars in a-d, 10μm; in a’-d’, 5 μm; in a”-d”, 1 μm.

Figure 6

Correlation analysis between the length of re-MaSp1 and mechanical properties of the silk fibres. The correlation analysis between mechanical properties, including maximum stress, maximum strain, Young’s modulus and toughness, and the number of the amino acids (aa) of re-MaSp1, respectively, are shown in (a–d). (a–d) The correlation analysis between mechanical properties and the length of re-MaSp1in heterozygous G4; (a’–d’) the correlation analysis between mechanical properties and the length of re-MaSp1 in homozygous G5; (a”–d”) the correlation analysis between mechanical properties and the length of re-MaSp1with similar protein levels in homozygous G5. Each dot represents the mean value of the fifteen pieces of silk fibres from each transgenic lineage. Note: Dots with different colours or sizes are used to distinguish close values for different transgenic lineages.

Figure 7

Schematic representation of synthetic spider silk gene and structural motifs. (a) The pUC57-MaSp1 vector including two repetitive units of re-MaSp1; (b) the pUC57-MaSp2 vector including three repetitive units of re-MaSp2; (c) the detailed amino acid sequence of one repetitive unit of re-MaSp1, Type1, Type2, Type3 and Type4 are the structural units derived from the black widow spider, L. hesperus; (d) the detailed amino acid sequence of one repetitive unit of re-MaSp1, Type1′ and 4′ are the structural units derived from the MaSp2 protein of the black widow spider, L. hesperus.