Cloning And Molecular Modification Of Baboon Uricase Gene

Uricase (UOX) is a key enzyme in the metabolic pathway of purine,which decides the final breakdown product of purine in living organism. Dueto the evolutionary fate that two non-sense mutations of uricase gene occurredin human genome, human can not produce uricase, which subsequently resultsin higher physiological concentration of uric acid in human body than in thosespecies who possess the active uricase gene. Excessive uric acid is consideredas the direct cause of diseases, such as hyperuricemia, gout, nephrolithiasis,and vascular diseases. Therefore, development of drugs for reducing uric acidhas keeping attracting interests from pharmaceutical industry and researchcycles. Despite the excellent performance on control of physiological uric acid,the drugs applied in clinic now, such as allopurinol, febuxostat andbenzbromarone have serious side effects, which actuates people to developmore effective and safer drugs. Uricase, as a living organism-derived enzyme,has a high efficiency on reduction of uric acid. However, as a heterogeneousprotein, it may trigger serious hypersensitive reactions when applied in humanbody. So, how to erase or decrease the immunogenicity of uricase becomes an issue that must be solved.This thesis aims at erasing or decreasing the immunogenicity of baboonuricase (bUOX) by way of modification of uricase with a proper, selectedmodifier. We took bovine lactoferricin (LfcinB) as a modifier and designedthree modified uricase fusions with LfcinB: rbUOX-LfcinB, rbUOX-LfcinB2,rbUOX-LfcinB3, in which rbUOX were connected by LfcinB at N-terminal,N-and C-terminal, and in molecule, respectively.Four genes encoding target proteins (rbUOX and three modified uricases)were first amplified by splicing by overlap extension PCR (SOE), andtransformed into proper E. coli host cell. After induction by IPTG, four geneshad expressed successfully the target proteins.By way of lowering induction temperature to25℃, the target proteinswere found in cytoplasm in a soluble form. After cell lysis, the target proteinswere separated effectively by Ni2+-His Bind Resin. The quantities of theexpressed proteins were detected by BCA method and results indicated thefields of the four proteins: rbUOX, rbUOX-LfcinB, rbUOX-LfcinB2, andrbUOX-LfcinB3were136.0mg/,127.5mg/L,116.6mg/L, and111.7mg/L,respectively. The recoveries of the proteins were over70%. Enzyme activitydetection on the acquired proteins revealed that the four proteins possessedactivities of17.93,8.10,6.23, and3.76U/mg, respectively. This result showedthat the activity of baboon uricase decreased after modified by LfcinB.For the characterization of the expressed proteins, MALDI-TOF-MS/MS was performed, and the results confirmed the amino acid sequences of the fourseparated proteins agreed very well with the theoretical proteins.As to the determination of immunogenicity of the modified rbUOX,rbUOX was first utilized to immune human leukocyte for7d to preparehuman anti-rbUOX antibody. And then a modified indirect ELISA wasoperated to detect the immunogenicity of modified uricases using rbUOX aspositive control and human serum albumin as negative control. Resultsrevealed that the immunogenicity of modified uricases declinedcorrespondingly with the amount of modifiers.Results presented here reveal it is helpful to decrease the immunogenicityof uricase by way of introduction of LfcinB and subsequently provide a novelstrategy on protein modification in enzyme engineering cycle.