Study On Refolding Human Deoxyribonuclease Ⅰ

Recombinant human deoxyribonuclease Ⅰ (rhDNase Ⅰ) is a specificDNA enzymes, which is composed of260amino acids. The monomerglycoprotein contains two disulfide keys and two potential N connectionglycosyl change site. DNase Ⅰ plays an important part in the occurrence ofsystemic lupus erythematosus, gastrointestinal tumors and myocardialinfarction. The commercial DNase Ⅰ have been used for the treatment ofsystemic lupus erythematosus and cystic fibrosis. DNase Ⅰ can get fromthe urine, however，the concentration is very low. RhDNase Ⅰ which isproduced by genetic engineering will have great advantage. The inclusionbodies of recombinant human deoxyribonuclease Ⅰ is researched in thispaper. First, the fermentation conditions of the gene engineering bacteriawere optimized in order to obtain large amounts of inclusion body;second, dilution refolding of inclusion body protein is studied; third,according to relevant data of the dilution refolding, three dialysisrefolding and three gel filtration chromatography refolding are researchedin order to improve inclusion body refolding effect. In37℃,bacterium which is inducted by the IPTG can express thetarget protein. The target protein exists in bacteria in the form of inclusionbody. The optimal fermentation conditions is that:37℃,IPTG is addedafter3h’s culture, medium is made from1.6%Try,1%YE,0.5%NaCl,2%inoculation amount,20%liquid volume,170r/min. In this way, theexpression of inclusion bodies accounted for27.7%of total bacterialproteins.0.16g inclusion bodies can be obtained (Dry weight).Recombinant human DNase I gene engineering bacterium arebroken by ultrasonic method. After centrifugal, the precipitation is theinclusion bodies of recombinant human DNase I. The inclusion bodycontains a large amount of impurities, which play a bad role in refolding.Impurities can be removed effectively by inclusion body washing liquid,after washing; inclusion bodies purity can reach90%. Under thecondition of4℃,2mol/L urea in refolding buffer，using pulsed injectinginclusion body denaturation solution，protein concentration is controledbetween100ug/mL and300ug/mL, which can get the best dilutionrefolding efficiency.Three dialysis refolding can get recombinant human DNase I whichowns biological activity. In principle, the three refolding dialysis canreduce the concentration of urea gradually to recovery the biologicalactivity of inclusion body protein, however, the specific operation isdifferent and the enzymatic activity, quality and rate of recovery and purity are different. In general, continuous dialysis method is better thanstep by step direct dialysis method better than dialysis.Three kinds of gel filtration chromatography can be used successfulin refolding of recombinant human DNase Ⅰ, and compared with thetraditional dilution refolding, Degeneration of protein can be utilized inhigh protein concentration, active recovery rate is higher, while therefolding proteins can be purification in some degree.Three types of gelchromatography refolding have the same principles and differentoperations, urea concentration gradient gel filtration chromatography andimprovement no urea concentration gradient gel filtrationchromatography are injected a urea concentration gradient in the gelchromatographic column upper to help inclusion body protein remove thedenaturing agent linearly, in order to realize refolding. In all, refoldingefficiency of urea gradient gel chromatographic is highest, andimprovement no urea concentration gradient gel chromatographic is alsovery good and the operation is relatively simple.