X-axis shows sequence number. research reagents in the future. Keywords:Antibody domain, CH2, Nucleolin, Yeast display, Monoclonal antibody, Aggregation prone region == 1. Introduction == Monoclonal antibodies (mAbs) have been highly successful as targeted therapeutic agents for diagnosing and treating diseases [1,2]. Despite their widespread application, it is widely accepted that full-size antibodies exhibit poor penetration into solid diseased tissues (such as solid tumor tissues) as well as inability to enter occluded regions of protein molecules due to their relatively large size (150 kDa) [35]. Protein scaffolds with relatively small size have distinct advantages because of greater and more rapid tissue accumulation and the ability to potentially target epitopes not accessible by full-size antibodies. Antibody-based small scaffolds include Fabs (60 kDa), single chain Fv fragments (scFvs) (2030 kDa) and domain antibodies (dAbs) (1215 kDa). Meanwhile, non-antibody-based scaffolds, such as fibronectin type III subunit, tenascin type III subunit, and ankyrin repeat proteins, are under development as novel therapeutics [68]. Through protein engineering technologies, these small scaffolds are being optimized for enhanced affinity, stability and expression levels [9]. Redesigned antibody-like scaffolds offer a novel class of therapeutics with Amoxicillin Sodium unique properties that are not possessed by full-size antibodies. They are promising as imaging reagents and candidate therapeutics. Human Ig constant 1 CH2 domain is an independent folding domain that comprises part of the Fc portion of Ig. Previous studies have proved that CH2 can be expressed and refolded as a soluble, monomeric domain at a high level [10]. CH2 domains can be engineered so as to retain some of the effectors functions that are not possessed by other smaller antibody-like Amoxicillin Sodium fragments. For example, isolated CH2 domains can confer full or partial functions of Fc receptor-binding and relatively long half-lives [11]. It was also proved that engineered CH2 exhibits remarkable thermostability with almost 20 C higher Tm than wild-type CH2 [10,12]. Importantly, CH2-based reagents are likely to be well tolerated in concentrations needed for achieving the long half-life, according to the fact that CH2 is a portion of all Ig subtypes, which exist in human whole blood with high concentrations [11]. Engineered CH2 domains are under development as scaffolds for construction of libraries containing diverse binders that provide novel candidate therapeutics Amoxicillin Sodium [10,13]. In recent years, several binders against the HIV-1 Env gp41 and gp120 have been successfully isolated from CH2-based phage libraries with diversified loop regions [14,15]. Yeast display is a powerful technology for isolating and engineering antibodies or proteins to increase their affinity, specificity and stability. Over past years, yeast display has emerged as an effective alternative to phage display technology and has particular advantages in the selection and affinity maturation of mAbs [1620]. In some cases, because the protein folding and secretory of yeast is similar to mammalian cells, it finds out more or different mAb or protein candidates SRSF2 rather than phage display [21,22]. Here, we constructed a large-size yeast display library based on a stabilized CH2 scaffold with CDR-grafting or mutagenesis of loops. Some binders were isolated from this yeast library by panning against a cancer-specific antigen nucleolin that is not selected using CH2 phage display libraries. In order to solve the aggregation issues, through potential aggregation prone region.