Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways

Hereditary tyrosinemia type 1 is an autosomal recessive metabolic disorder, which is caused by a defective fumarylacetoacetate hydrolase enzyme, and consequently metabolites such as succinylacetone and p-hydroxyphenylpyruvate accumulate. We used a modified comet assay to determine the effect of these metabolites on base- and nucleotide excision repair pathways. Our results indicate that the metabolites affected the repair mechanisms differently, since the metabolites had a bigger detrimental effect on BER than on NER.     

Efficient Recovery of High-Affinity Antibodies from a Single-Chain Fab Yeast Display Library

Yeast display is a powerful technology for the isolation of monoclonal antibodies (mAbs) against a target antigen. Antibody libraries have been displayed on the surface of yeast as both single-chain variable fragment (scFv) and antigen binding fragment (Fab). Here, we combine these two formats to display well-characterized mAbs as single-chain Fabs (scFabs) on the surface of yeast and construct the first scFab yeast display antibody library. When expressed on the surface of yeast, two out of three anti-human immunodeficiency virus (HIV)-1 mAbs bound with higher affinity as scFabs than scFvs. Also, the soluble scFab preparations exhibited binding and neutralization profiles comparable to that of the corresponding Fab fragments. Display of an immune HIV-1 scFab library on the surface of yeast, followed by rounds of sorting against HIV-1 gp120, allowed for the selection of 13 antigen-specific clones. When the same cDNA was used to construct the library in an scFv format, a similar number but a lower affinity set of clones were selected. Based on these results, yeast-displayed scFab libraries can be constructed and selected with high efficiency, characterized without the need for a reformatting step, and used to isolate higher-affinity antibodies than scFv libraries.