**2. Cysteine-stabilized** α**/**β **motif**

## **2.1 Specific pattern and structural feature**

CSαβ motif is a very unique protien scaffold. Proteins with a CSαβ motif express a high diversity in their protein primary structures (Figure 1) but share a common core sturcture that consists of a α-helix and an anti-parallel triple-stranded β-sheet (Figure 2 a to d). This is especially interesting not only for academic research but also very useful for applied utilities. From the protein sequence alignment analysis, six cysteines and one glycine (-C-X*l*-CXXXC-X*m*-GXC-X*n*-CXC-) are exzactly conserved in all proteins containing a CSαβ motif (Carvalho & Gomes, 2009;Lay & Anderson, 2005). The cysteines form a framework and tightly connect the intramolecular structures. The most significant feature of the framework is two disulphide bonds which are formed with a patten of (*i, i+4*) and (*j, j+1*). The cysteines of (*i, i+4*) and (*j, j+1*) are located in the α-helix and the β3 strand of the β sheet; respectively (Figure 1). The helix and the sheet are connected by the disulphide bonds pairing with 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 &

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

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

CSαβ motif is a very unique protien scaffold. Proteins with a CSαβ motif express a high diversity in their protein primary structures (Figure 1) but share a common core sturcture that consists of a α-helix and an anti-parallel triple-stranded β-sheet (Figure 2 a to d). This is especially interesting not only for academic research but also very useful for applied utilities. From the protein sequence alignment analysis, six cysteines and one glycine (-C-X*l*-CXXXC-X*m*-GXC-X*n*-CXC-) are exzactly conserved in all proteins containing a CSαβ motif (Carvalho & Gomes, 2009;Lay & Anderson, 2005). The cysteines form a framework and tightly connect the intramolecular structures. The most significant feature of the framework is two disulphide bonds which are formed with a patten of (*i, i+4*) and (*j, j+1*). The cysteines of (*i, i+4*) and (*j, j+1*) are located in the α-helix and the β3 strand of the β sheet; respectively (Figure 1). The helix and the sheet are connected by the disulphide bonds pairing with the

d'Anjou, 2009).

based on the scaffold.

**2. Cysteine-stabilized** α**/**β **motif** 

**2.1 Specific pattern and structural feature** 

functions and stabilities of a protein scaffold.

pattern (*i, i+4*) and (*j, j+1*) (Assadi-Porter et al., 2000;Fant et al., 1999;Sun et al., 2002;Zasloff, 2002;Zhu et al., 2005). The third disulphide bond connects loop 1 and and β2 strand. In the β2 strand one glycin is conserved and locates in the central region of the motif. In plant defensins, there is usually a forth disulphide bond that seal the N- and C- terminee. The positively charged α-helix is another character. The positively charged residues are especially concentrated in the helix. β-sheet of the motif is formed by a hydrophobic amino acid clust and the sheet is composed by two or three anti-parallal strands. Loop regions of the mofit are highly diversed in lenght among proteins. There is a small turn that connects the helix and β2 strand.

Fig. 1. The sequence alignment of proteins containing CSαβ motif. Sequences of two scorpion toxins (UniProt P13487, P0C5H3), two plant defensins (UniProt P56552, B6DX36), two insect defensins (UniProt P10891, Q38LE8) and two mollusk defensins (UniProt P85008, C4NY93) are aligned. The restrictly conservered residures, include six cysteins and one glycin, are high lighted in red. The green arrow box, red box and cyan boxs above the alignment represent β strands, α-helix and loops; respectively. The blue lines above on the top indcate the disulphide bond bwteen cysteines.
