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The success of macromolecular crystallization depends on the protein's ability to form specific, cohesive intermolecular interactions that serve as crystal contacts. In the cases where the protein lacks surface patches conducive to such interactions, crystallization may not occur. However, it is possible to enhance the likelihood of crystallization by engineering such patches through site-directed mutagenesis, targeting specifically residues with high side chain entropy and replacing them with small amino acids (i.e., surface entropy reduction, SER). This method has proven successful in hundreds of crystallographic analyses of proteins otherwise recalcitrant to crystallization. Three representative cases of the application of the SER strategy, assisted by the automated prediction of the mutation sites using the SER prediction (SERp) server are described. [ABSTRACT FROM AUTHOR]

Engineered polypeptides have emerged as attractive materials to construct artificial extracellular matrices (ECMs) in tissue engineering. These materials offer advantages over conventional synthetic materials in recapitulating essential characteristics of complex and dynamic native ECMs, which is one of the key requirements for successful tissue engineering, because proteins are major players in providing structural support, cell adhesion, and signal regulation in native ECMs. The structures and functions of these proteins and their domains, as well as those of de novo designed polypeptide domains having self-assembly and molecular recognition abilities, can be combined in engineered polypeptides in a modular manner to yield multifunctional, bioactive materials to mimic native ECMs and optimize tissue engineering outcomes. Engineered polypeptides can be synthesized both chemically and biosynthetically. In recent years, the biosynthetic methodology has received increasing attention because the advances in molecular biology and protein engineering have expanded its capacity. Biosynthetic preparation allows polypeptide materials to be genetically engineered in a modular manner and the resulting polymers have absolutely uniform sequence, composition, molecular weight, and consequently higher order structures and functions. These properties not only allow us to engineer novel multifunctional materials to elicit desired cell responses toward functional tissue regeneration, but also offer the opportunity to create well-controlled and tunable systems for systematic studies to enhance our understanding of the relationships among extracellular microenvironments, cell behavior and fate selection, and tissue assembly. Such understanding will provide valuable guidelines for design of future generations of artificial ECMs. In this chapter, engineered polypeptides that have been used or have the potential to be used in tissue engineering will be discussed with an emphasis placed on their molecular design as well as examples of their use in tissue engineering studies. [ABSTRACT FROM AUTHOR]

Protein splicing elements, termed inteins, were first identified in 1990. Since then, post-translational protein splicing has been demonstrated and the selfcatalytic mechanism deciphered. The robust nature of these single turnover enzymes is evidenced by the expanding list of naturally occurring variations in the protein splicing mechanism. Protein splicing must be efficient and neutral, and must not cause detrimental effects to the spliced extein; otherwise, selective pressure would lead to intein loss. Inteins are probably ancient elements, but their original function can only be speculated upon, because invasion by homing endonucleases mobilized them into new locations and converted them into selfish DNA. To date, there is no evidence of regulation of protein splicing in native systems. The sporadic distribution of inteins may relate more to the types of genes found in mobile elements capable of spreading inteins, than to the function of those genes. Inteins tend to be found in conserved host protein motifs, which may be due to conservation of homing endonuclease recognition sites, difficulty in removing inteins from essential regions or the ease of accepting an insertion sequence in a conserved substrate or cofactor binding site designed to interact with the environment. The ability to cleave peptide bonds, to ligate protein fragments and to generate carboxy-terminal alpha-thioesters have made inteins the fastest growing tool for protein engineering and biotechnology. [ABSTRACT FROM AUTHOR]

Intein protein-splicing domains are part of the Hint superfamily. This superfamily includes three other characterized families: Hog-Hint and two types of Bacterial intein-like (BIL) domains. Hint domains share the same structure fold and common sequence features, and have similar biochemical activities. They post-translationally auto-process the proteins in which they are present by protein-splicing, self-cleavage or ligation activities. Yet, each Hint family apparently has its own distinct biological role. We discuss the evolution of the different Hint families, the origin of primordial Hint domains themselves, and their possible activities and biological functions. [ABSTRACT FROM AUTHOR]

An abstract of the article "Engineering, expression, purification, and production of recombinant thermolysin," by Kuniyo Inouye is presented.

A definition of the term "knockout animal" is presented. It refers to an animal in which genetic code for a protein was removed. It is also known as transgenic animals.