Anti-6xHIS Tag polyclonal antibody

His-tagged molecular species, a ferrocene derivative and Protein A, were immobilized on electrode surfaces (Au and graphite) through formation of a chelated complex in the presence of Cu2+ cations used as bridging units. The complex was cleaved and the attached molecules were released from the electrode surface by applying reductive potential to the electrodes resulting in Cu2+ reduction, thus decomposing the chelate complex. The molecule release process was followed by cyclic voltammetry in case of the ferrocene derivative. His-tagged Protein A was additionally labeled with a fluorescent tag and its release was followed by fluorescence measurements in the solution and by impedance spectroscopy at the electrode. The studied release of the His-tagged redox species and biomolecules was considered as a new generic approach to the signal-controlled molecule release applicable in various biotechnological and biomedical applications.

Hispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6 x His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell factory. Results showed that the E. coliderived recombinant Hispidalin did not show any antimicrobial activity against all the tested strains, whereas the P. pastoris-derived recombinant Hispidalin (rHispidalin) showed a broad antibacterial spectrum against five pathogenic bacteria of both Gram-negative and Gram-positive. rHispidalin also has bactericidal activity and completely killed all of the Staphylococcus aureus within 40 min. Additionally, rHispidalin showed a broad range of thermostability and pH stability, and a hemolytic activity of less than 2% even at a concentration of 300 mu g/ml; it was resistant to trypsin and proteinase K, but was moderately sensitive to pepsin and papain. Moreover, rHispidalin effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. After an initial optimization was performed, the amount of rHispidalin accumulation could reach as high as 98.6 mu g/ml. These results indicate that Hispidalin could be produced on a large scale by P. pastoris and has a great potential to be utilized as a new antibacterial agent for further development.