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Antibody mimetics are proteins smaller than antibodies that have antigen-binding properties. Here, we used a combination of informatic selection and experimental verification to identify from a public database several candidate scaffold proteins. From those candidates, we selected a protein with high thermal stability, bacterial expression, and mutation tolerance and used molecular engineering and phage display selection techniques to develop the scaffold into an antibody mimetic with binding affinity for galactin-3, a biomarker of cancer and heart disease. [ABSTRACT FROM AUTHOR]

The interaction between biomolecules and inorganic materials has gained much interest for the development of sensors and highly controlled materials in scale and composition. For this purpose, 4F2, a VHH antibody with high affinity and specificity toward zinc oxide (ZnO) surfaces has been constructed. Here, we report that 4F2 also has an affinity toward zinc ion (Zn2+). The stability of 4F2 and its Zn2+ complexes was determined using differential scanning calorimetry. We then determined the binding affinity between 4F2 and Zn2+ by using isothermal calorimetry. To further elucidate the molecular binding mechanism of 4F2 toward Zn2+, we applied the ¹H-15N heteronuclear single quantum coherence NMR technique. We found that 4F2 recognized Zn2+ near the complementarity determining region 3 (CDR3). Our results provide the basis for the recognition mechanism of ZnO by 4F2. [ABSTRACT FROM AUTHOR]

A mixture of protein-encoding gene fragments was ligated into linearized expression vectors in one pot at a time (iPaT) to simultaneously generate each protein expression vector. This high-throughput iPaT ligation method was stable and highly efficient for the simultaneous generation of more than 10 different expression vectors. [ABSTRACT FROM AUTHOR]

Kinesin is one of the numerous motor proteins present in the cell. Its function is to transport cargo along microtubules, which serve as tracks, by efficiently making use of chemical energy. Here, we have studied the translocation of quantum dots by kinesin. Kinesin was bacterially expressed and conjugated to quantum dots, thus allowing observation of the motion of the quantum dots along the microtubules by fluorescence microscopy. The velocity was determined by a kymograph, and was in agreement with expectations. [ABSTRACT FROM AUTHOR]