| Acinetobacterbaumannii is a human pathogen that is becoming increasinglywellknown inboth theclinical and scientificfields. This gram-negative bacteriumwas discovered to have amorphous properties and has become a topic of concern in many clinical settings. Theseconcernsinclude:the ability to formbiofilms, which increases itsresistance to therapeutic drugs;its high tolerance to various environments;and its ability to survive on multiple surfaces.The goal of this project was to characterize,clone,and purifya secretedserine protease present in A. baumannii called Prolyl Oligopeptidase FamilyProtein 2 (POP 2).We identified this protein due to its similarity to other proteases. Further confirmation of this protease was done using automated prediction programs. Once the gene was identified,it was cloned from A. baumanniigenomic DNA usingPCR,and subclonedinto thepET100 cloning vector. The ligated vector was transformed and propagated in E. coliTOP10 cells.Confirmed clones were transformed intheE. coliexpression strain,BL-21.The protein expression was accomplished using an auto-inductionmethodwhich produced more protein than other methods. Successful expression of the POP2 protein allowed forthe purification processusing Immobilized Metal Affinity Chromatography (IMAC). All processes completed throughout this project help in understanding how A. baumannii uses POP2against host defenses.Results of this researchcan possibly lead to future advancementsin targeted drug therapiesagainst A. baumannii infection.KEY WORDS: Acinetobacter baumannii, Prolyl Oligopeptidase Family Protein(POP2), cloning, protease, protein, purification, peptidase |