The wide variety and structural properties of natural spider proteins provide design flexibility for constructing recombinant spider silk proteins, as their composition, structure, and degradability can be adjusted. By incorporating a natural or artificial gene encoding the target protein into plasmid DNA, the plasmid gene construct is introduced into the host for expression (most commonly in E. coli). The artificial gene's expression produces the desired recombinant spider silk protein, which is ultimately extracted and purified from the host, resulting in the desired functional protein.
Although the functions of spider silk vary, the different spider silk protein structures all consist of three parts, namely the non-repetitive N-terminal structural domain (NT), the intermediate repetitive structural domain (REP), and the non-repetitive C-terminal structural domain (CT). To obtain high-performance artificial spider silk protein materials, several recombinant spider silk proteins have been designed and produced. However, these recombinant proteins usually contain only one or more repetitive modules or have N-terminal and/or C-terminal structural domains in addition to the repetitive modules.
There are often several ideas in designing recombinant protein sequences using genetic engineering: (1) selection of specific structural domains and their combinatorial design; (2) heterozygous design; (3) sequence functionalization; (4) generation of dynamic stimulus response systems; and (5) introduction of non-natural amino acids.
The protein was constructed by a "Head-to-tail" gene construction strategy. The clone construction screening process was performed in E. coli strains, and the positive clones were screened by enzyme digestion to obtain plasmids with the correct insert length. They were sequenced, and the correctness of the gene sequence was identified by nucleotide gene sequencing.
Protein size, isoelectric point, absorbance, and other information, as well as the affinity of the constituent amino acids, were analyzed and predicted using the online tool on the ExPASy website.
Transform the plasmids into E. coli strains and culture them under certain conditions. Centrifugation was performed, and the bacteriophage was collected. And then, the mixture of cells and culture medium is broken using a cell crusher. The supernatant containing the target protein is collected by centrifugation. The purity of the protein is determined, and the protein size is characterized, and the specific expression of the protein is verified using Western blot.
We have several methods to prepare spider fibers, so you can ask us for the most suitable preparation route.
Analysis and testing can be performed by circular dichroism, and Fourier infrared spectroscopy.
Scanning electron microscopy can be used to take pictures and measure the diameter of the fibers.
Mechanical properties are tested using a nano-sensing tensiometer, using software to record and calculate property parameters such as tensile strength at break, elastic tensile, Young's modulus, and toughness of silk fibers.
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Lifeasible has established a one-stop service platform for plants. In addition to obtaining customized solutions for plant genetic engineering, customers can also conduct follow-up analysis and research on plants through our analysis platform. The analytical services we provide include but are not limited to the following:
July 13, 2024
