Molecular Biology Methods to Construct Recombinant Fibrous Protein.

Recombinant technologies are often used to synthesize fibrous proteins that are difficult to separate and extract in nature, such as spider silks and elastin. Although the recombination techniques can be diverse, PCR, gel electrophoresis, and seamless cloning, as the basic methods of molecular biology, have been widely used for constructing fibrous proteins' homologous recombinant plasmids. Considering that some readers of this book may not have a molecular biology background, in this chapter, we will introduce these three most used and effective recombination techniques. For PCR, we primarily introduce colony PCR, high-fidelity PCR, and overlap PCR, which are three kinds of the most used methods. In terms of seamless cloning, the detailed protocols of Gibson Assembly and Golden Gate Assembly are introduced. The introduction of this chapter is expected to provide a comprehensive methodological reference for the following chapters to introduce the recombination of specific fibroin proteins.

Spinning Methods Used for Construction of One- and Two-Dimensional Fibrous Protein Materials.

Natural silk protein fibers have shown a great attraction to the researchers due to the extraordinary mechanical property, biocompatibility, and functional diversity. Unfortunately, the low yield and unevenness have hampered the scale use of the natural silk fibers. Herein, the appearance of the bioinspired artificial spinning strategy offers an effective way to fabricate silk fibers with controllable structures and functionality. This chapter describes an experimental method to prepare silk protein fibers on a large scale and summarizes the method to investigate the effects of the structure-property relationship of the recombinant protein fibers.

Gelation Methods to Assemble Fibrous Proteins.

Gelation is an efficient way to fabricate fibrous protein materials. Briefly, it is an aggregation process where protein molecules assembly from a random structure into an organized structure such as nanofibrillar networks. According to their mechanisms, the fibrous proteins gelation can be classified into physical gelation and chemical gelation. The physical gelation is formed by the conformational transformation of fibroin proteins, which can be triggered by temperature, concentration, pH, or shear force. On the other hand, the chemical gelation is to cross-link fibrous proteins through chemical and/or enzymatic reactions. In this chapter, we summarize the protocols for preparing fibrous protein hydrogels, including both physical and chemical methods. The mechanisms of these gelation methods are also highlighted.

Assessing the homogeneity of the elastic properties and composition of the pig aortic media.

Most previous studies of arterial wall elasticity and rheology have assumed that the properties of the wall are uniform across the thickness of the media and, therefore, that the relationship between stress and strain may be described by a constitutive equation based on a single strain energy function. The few studies where this assumption has been questioned, focussed on differences between the adventitia and the media rather than on differences within the media itself. Here, we report in vitro elasticity and residual strain measurements performed separately on the inner and outer half of the pig aortic media, together with a histomorphometric assessment of the radial distribution of elastin, collagen and smooth muscle cell numbers. Although we found that the pressure-diameter relationships of the two halves were dissimilar, when allowance was made for their different unloaded dimensions, their material properties agreed closely, a result in keeping with the observed uniform radial distribution of scleroprotein and vascular smooth muscle. We also found a difference in the opening angle (which is often taken as a measure of residual strain) between the inner and outer medial halves. However, strain analysis showed that the opening angle is an extremely sensitive measure of residual strain and that the difference in the actual magnitudes of residual strain between the two halves of the media was small. We conclude that the media of the porcine thoracic aorta has similar elastic properties throughout its thickness and that this uniformity is matched by a uniform distribution of matrix protein and vascular smooth muscle cells. Furthermore, the distribution of strain in the media can adequately be described by a single-layer model with uniform elastic properties throughout its thickness.