Electrospining Machine: Efficient biosynthetic preparation of spider silk with high spinning performance

Background

Flexible wearable piezoelectric sensors, with their remarkable ability to convert small deformations into electrical energy, have emerged as highly promising technological solutions with numerous potential applications in healthcare monitoring, human-machine interfaces, biomechanics and smart motion science, as well as security and surveillance. Nanofiber mats consist of interconnected nanoscale fibers, typically ranging from hundreds of nanometers to submicrometers in diameter, which have extraordinary responsiveness to mechanical stimuli such as pressure, strain, or vibration, and are perfectly flexible and inherently mechanically strong, making them advantageous components for building piezoelectric sensors. These pads can be fabricated by a variety of techniques, including electrospinning, solution blow-spinning, and others..

The main point of this paper

Importance of Synthetic Spider Silk Electrospun Nanofbers:

Spider silk has great potential for application in various fields such as biomedicine and tissue engineering due to its excellent mechanical properties and biocompatibility

Strategies to improve the efficiency of MaSp heterologous expression:

Researchers have developed several strategies to improve the heterologous expression efficiency of the major Ampulla spider silk protein (MaSp). One of these approaches is to design minispiroplasma proteins, which consist of an end structural domain and a central repeat region that are significantly shorter compared to natural spiroplasma protein templates

To improve silk-forming ability, the researchers replaced the alanine motif in the central repeat region of MaSp with an amyloid-like peptide and increased the number of central repeat regions of MaSp, resulting in ultra-high molecular weight recombinant spider proteins

Challenges of recombinant spider proteins:

The high GC content of natural spider silk genes, the highly repetitive amino acid sequences in the repetitive core region, the high specific amino acid content, and the high molecular weight pose a great challenge to their efficient heterologous expression, which limits the wide application of spider silks

Optimization of MaSp sequence and expression strategy:

Optimized design of the sequence of recombinant spider silk proteins, combined with heterologous expression strategies, has greatly facilitated the biosynthesis of multifunctional spider silk proteins

Using metabolic engineering, the Xia Xia-Qian Zhigang joint research team dramatically increased the yield of high molecular weight recombinant arachnid proteins synthesized intracellularly in Escherichia coli by increasing the supply of glycyl-tRNA and employing a low-temperature-induced expression strategy, reaching 3.6 g/L

Challenges and progress in industrialized production:

In order to simplify the protein purification process, the group developed an extracellular secretion platform of arachnid proteins from Corynebacterium glutamicum for the first time, and realized the production of highly water-soluble recombinant arachnid proteins, which lays the foundation for industrial production

Construction of amyloid chimeric arachnid proteins:

Based on the computer prediction of spatial zipper conformation, we synthesized artificial mini-arachnid proteins by introducing self-designed amyloid hexapeptide sequences with high propensity to form spatial zipper conformation, and obtained artificial spider silks with higher tensile strength and toughness comparable to that of natural traction silks

Electrospinning preparation of bionic spider silk protein Amy-6rep and its application in nanogenerators

Spider silk protein properties and challenges:

Spider silk protein (spidroin) has a unique three-dimensional structure that determines its excellent mechanical properties. However, the difficulty of heterologous expression and poor spinning properties of recombinant spider silk proteins limit their application

Mimetic spider silk protein (Amy-6rep) was developed:

Through sequence modification, researchers obtained a high-yield, low-molecular-weight bionic spider silk protein (Amy-6rep), which increased the highly fibrillated microcrystalline regions in the core repeat region of natural spider silk proteins and replaced the polyalanine sequence with an amyloid polypeptide

Amy-6rep expression and self-assembly properties:

As a result of the sequence modification, the expression of Amy-6rep was increased by approximately 200% and the self-assembly properties were significantly enhanced

Application of electrospinning technology:

Nanofibers prepared by electrospinning technology using Amy-6rep exhibited good friction power generation ability, verifying its excellent spinning performance

Validation of nanogenerators:

The use of Amy-6rep as a nanogenerator validates its potential application in electrostatic spinning technology and provides a new strategy for the production of artificial nanogenerators

Significance of the research results:

This research not only improves the understanding of spider silk protein production, but also provides valuable insights into the design and optimization of PVDF nanofibers with tunable nanofiber size, alignment, and significant piezoelectric properties, with important applications in the field of high-performance fiber materials and nanotechnology.

Summarize

Here, we combined the low molecular weight spirulina strategy with the sequence modification strategy of amyloid polypeptide substitution based on the similarity of the β-folded crystal structure, and proposed the ideal number of microcrystalline regions, which increased the number of highly fibrillar microcrystalline regions in the core repeat region of the natural spirulina to six, and limited the sequence length. Finally, by constructing a heterologous expression vector in Escherichia coli, the high yield and low molecular weight of bionic spiropodophyllin was obtained. Due to the low sequence repeat rate of the amyloid polypeptide in the bionic spiropodin, the heterologous expression yield of the bionic spiropodin Amy-6rep was increased by ≈200%, which laid the foundation for the subsequent large-scale spinning. A reasonable increase in the number of microcrystalline regions proved to improve the self-assembly performance of the bionic spiroprotein, but had no significant effect on the heterologous expression level. Notably, we used a simple inclusion body washing method for bionic spirochete proteins expressed in precipitated form, and the results showed that the recovery of this purification method reached ≈80%, which significantly improved the recovery efficiency compared with the previous Ni-NTA purification method and indirectly increased the yield of bionic spirochete proteins.