**1. Introduction**

628 Biomedical Science, Engineering and Technology

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> Protein engineering is a process that modifies/creates functions or increases stabilities of proteins through artificial selection and evolution (Angeletti, 1998). For the fast development of molecular biology techniques, numbers of proteins have been successfully engineered to equip noval functions in the past several decades (Alahuhta et al., 2008;Bottcher & Bornscheuer, ;Ehren et al., 2008;Huston et al., 1988;Leta Aboye et al., 2008). These engineered proteins are developed either for academic research purposes or biomedical applications. The core of protein engineering is an appropriate protein scaffold (Hey et al., 2005;Pessi et al., 1993;Skerra, 2007). An excellent protein scaffold not only provides a platform for developing noval functions but also has many benefits, such as cost reduction during development/production or lasting efficacy of protein. An suitable protein scaffold should equip several characteristics (Hey et al., 2005;Pessi et al., 1993;Skerra, 2007). To introduce tailored functions into protein scaffolds is a major challenge and has a unique significance in protein design (Hey et al., 2005;Pessi et al., 1993).

> A protein scaffold is a peptide framework that exhibits a high tolerance of its fold for modifications (Hey et al., 2005). Candidates for suitable protein scaffolds should exhibit a compact and structurally rigid core. The folding properties of the protein scaffolds should not be significantly changed when the side chains in a contiguous surface region are replaced or loops of varying sequence and length are presented (Skerra, 2007). Several additional priorities have to be considered if the scaffold is used in biomedical applications. The scaffold must display extra stabilities to environments, such as low pH, high concentration of chaotropic agents and high temperatures. Molecular weight of the scaffold should be low and small molecules have advantages in passing tissue barrier (Baines & Colas, 2006). The scaffold should highly resist protease degradation and this ensures the engineered proteins can be safe in the gastrointestinal tract and not degraded (Aharoni et al., 2005). Low immunogenecity is important to reduce unexpected side effects and damage of healthy tissue (Van Walle et al., 2007). Post-translational modification of protein is an important issue, too. Most of eukarytic proteins require posttranslationally modified then gain their functions. These modifications includ glycosylation, cleavage of pre-peptide, formation of disulphide bridge and association of multiple peptides. Appropriate glycosylation also could reduce immunogenicity of proteins (Kosloski et al., 2009;Wang et al., 2010;Wu et al.). Currently, bacterial cells are the

most conventional and convenious tools for mass production of recombinant proteins, but it is not easy for bacterial cells to undergo post-translational modication of eukaryotic proteins (Jacobs & Callewaert, 2009;Muir et al., 2009). Even expressed in eukaryotic cells, glycosylation of proteins is not exactly the same among difference specises (Perego et al., 2010). Unappropriatly modified recombinant proteins might lead to unexpected immune respones, if the proteins are used for medical purpose (Kosloski et al., 2009; Li & d'Anjou, 2009).

Protein designing challenges our understanding of the principles underlying protein structure and is also a good method to access our understanding of sequence-structure and structure-function relationship (Nikkhah et al., 2006). Rational design of proteins requires detailed knowledge of protein folding, structure, function, and dynamics (Chen et al., 2005). To build expression libraries, it is necessary to understand a protein scaffold in detail to amino acids usage on each residue position. This would reveal the key elements that affect functions and stabilities of a protein scaffold.

The appearance of new intellectual property, the breakthroughs in technology, or the increase in a market need are three major impacts to biopharma industry. Protein engineering is a branch of the biopharm industry, where intellectual property rights apparently play the most important role in the development and commercialization of final products. The intellectual property strategy to protect inventions of biopharma industry is to patent and to license them on an exclusive basis. The intellectual property also must be included in the linkage of research/development and business to ensure the commercial viability of biopharma products and to cope with the rapid changes of market. Currently, the intellectual property situation of biopharma industry is too complex and hampers the generation and production of recombinant protein/peptide drugs. Pantent analysis and patent map are necessary and helpful while planing the research and development and marketing. A well planed intellectual property strategy can not only protect the output of research and development but also defend market. A protein scaffold with simple legal situation will avoid knotty lawsuits. In recent years, shouts for alternative protein scaffolds is urgent and alternative protein scaffolds usually provide a favourable intellectual property situation. In this chapter, we will focus on a protein scaffold, cysteine-stabilized α/β (CSαβ) motif, and discuss protein engineering based on the scaffold.
