Study On Recombinant Human KD/APP Variant

Serine protease are a diverse group of enzymes that exist in biologicalbody and keep an equilibrium between serine proteases and their specificinhibitors.The proteases inhibitor are divided by four types by their differentreactive centers in which the serine proteases inhibitors play a important rolein modulating physiological process ,such as coagulation, fibrinolysis,inflammatory respone,cell migration cell differentiation, complementactivation and release of hormone of peptide and protein.The catalysis centerof serine proteases have a active specific serine side chain which the active siteof protease inhibitor occupied the S1 position and formed complexes ofenzyme –substrate.The serine proteases inhibitor was divided 13 familiesaccording to their homologous sequence ,topostructure of disulfide bond andmechanism of function.The bovine Pancreatic Typsin inhibitor is arepresentative inhibitor of Kunitz family.BPTI extracted from bovine pancreasis serine protease inhibitor which has 58 amino acids with three disulfidebridges.It inhibits trypsin with 1:1 stoichiometry.BPTI is a nonspecific andbroad-spectrum serine protease inhibitor which affects known serine proteasessuch as trypsin,chymotrypsin,plasmin and kallikrein. It can defend bloodplatelet against bleeding, treat the patient with cruor impediment, in whichsituation the hemorrhage may occur. BPTI can be used in cardiac surgery asbeing extracorporal circulated to decrease the incidence of bleeding and theinflammatory reaction caused by the surgery. But infusion of high doses ofthese nonmammalian inhibitor could result in immunologic reactions inpatients undergoing repeat use.So we need find a substitute which ispreferably ,small ,potent and of human sequence origin.The amyloid β-proteinprecursor contains a domain homologous to Kunitz-type serine proteaseinhibitors.The sequence of KD/APPwhich is consisted of 57 amino acids has43% sequence identity with that of BPTI. The tertiary fold ,reactive center andmechanism of function of KD/APP is very similar to that of the Kunitzinhibitor BPTI.The KPI containing isoforms (APPIs)show ability to inhibit avariety of serine proteases such as factor XIa,epidermal growthfactor(EGF)-binding protein,the γ-subunit of nerve growth factor,expeciallythe inhibition constant Ki of KD/APP to factor XIa is higher than BPTI. TheAPPIs has growth factor activity and is an effective cell adhesionmolecule.they probably plays a role in the complex series of events that lead totissue repair at vascular wound sites and protect neurocytes against injury.Thepathological and physiological role of this protease inhibitor domain whichconcerns with abnomal metabolism of APP are not well understand.So weneed to produce large quantity of rhKD/APP to discover and study the possiblemechanism on pharmacodynamics.In order to improve these situations, studies were done as follows:① we constuct expression vector pPICZα-KD200 and transform it intoPichia pastoris via electroporation. pPICZα-KD200 secreted into the culturesupernatant with high level by the Pichia pastoris was screened. ② TheKPI/AβPP was expressed in 80L fermentor with optimizing parameters onlarge scale and the broth was harvested and purified with cation exchangechromatography. ③ Observation of the effect of rhKD/APP on primarilycultured hepatocytes at the cell level.④ Observation of protective effects ofrhKPI/AβPP on acute hepatic injury and experimental chronic liver injuryinduced by carbon tetrachloride on and and inhibit in rats in vivo.1. Construction of Pichia pastoris expression system for rhKPI/AβPP.The rhKPI/AβPP. expression vector pPICZα-KD200 was constructedby performing PCR with specific primer with Xho I and Xba I target sitesand a part of α-mating factor signal peptite.After identification byendonuclease digestation assay and sequencing, inearized expression vectorpPICZα-KD200 was transformed into Pichia pastoris via electroporation.The transformed Pichia pastoris were cultured in medium with zeocin resistantfor 28oC,36h .The transformed Pichia pastoris were screened and the genomicDNA of the screened yeasts were extracted using PCR technique. Yeast clonestested positively by PCR was Proliferated and then the expression ofrhKD/APP was induced with 0.5%methanol. The supernatant of fermentationis identified by SDS-PAGE, inhibition activity to trypsin and purified bycation exchenge and hydrophobic chromatography. The results showed thatrhKD/APP was identical with the native rhKD/APP in molecular and physicsand chemical properties.2. Studies on large-scale fermentation and purification process of rhKD/APP:(1)Studies on large-scale fermentation process of rhKD/APP: Pichiapastoris has many advantages as a kind of expression host, and it's verysuitable for large-scale expression of the extraneous proteins. So afteridentifying the bioactivities of the rhKD/APP, we explored the large-scalefermentation process of rhKD/APP and found that the best pH is pH3.3±0.2,DO between 25%～30% and the supply speed of methanol is 8.5ml/h/L initialfermentation volume. The concentration of rhKD/APP in the broth can reached1.0g·L-1.(2). A new method to purify rhKD/APP at large-scale: The supernatant offermentation was diluted with deionized water and adjusted to pH3.8 bysodium hydroxide .The diluted broth was loaded onto column of SP SepharoseFF cation exchange resin .The absorded rhKD/APP was eluted by 1mol·L-1NaCl and collected at 280nm.We found that this method is simply and reducedcost with the degree of purity being more than 95% and the yield coefficientwas higher than 70%.3.The effect of rhDK/APP on survival of rat hepatocytesThe purified rhKD/APP inhibits a variety of serine proteases in vitro. Inorder to find the biologic function of rhKD/APP as a protease inhibitor fromhuman origin at the cell level, it was added into rat hepatocytes in primaryculture for 96 hours in the medium supplemented with 2% new born calfserum. In contrast ,the medium containing 2%, 8% new born calf serum and10 μg·ml-1 BPTI respectively were used under the same conditions statedabove. It was found that rhKD/APP has the effect on increasing the survival ofrat hepatocytes by observing the morphological feature and cell growth curveanalysis,and still maintained the hepatocyte-like structure. The purifiedrhKD/APP was effective at 2.5 μg·ml-1 and maximally effective at 10 μg·ml-1.These results indicated that the rhKD/APP prolongs the survival of rathepatocytes.4.Hepatoprotective effects of rhKD/APP on acute hepatic injuryTo observe the protective effects of rhKD/APP on carbontetrachloride-induced acute hepatic injury and find possible mechanisms ofhepatic injury, Pathologic model of acute hepatic injury in mice caused bycarbon tetrachloride was set up and rhKD/APP with different dosage(4.5,9,18mg?kg-1) were administrated (ip) to mice at daily.on 7 day. rats were injectedwith CCL4at a dose of 10 mg?kg-1 body weight as a 0.2% vegetal oil solutionand control ones with the same dose of vegetal oil 1h after the last injection ofrhKPI/AβPP . All animals were starved overnight after CCL4 treatment andkilled 1h later after the injection the rhKPI/AβPP again.Blood was collectedfrom the femoral artery under light ether anaesthesia.Serum was separated bycentrifugation for the assay of alanine aminotransferase (ALT) and aspartatetransferase(AST)activities.For light microscopy,part of the liver tissue wasfixed in 10% buffered formalin ,embedded in paraffin wax ,and stained withhematoxylin and eosin. The mice treated with rhKD/APP exhibited the amountof ALT,AST in blood serum and MAD in hepatic tissue were decreased andhepatic tissue damage was improved .The rhKD/APP preventive effects onhepatic injury showed linear relation to the drug dosage.5.protective effect of rhKD/APP on chronic hepatic injuryTo observe the protective effects of rhKD/APP on carbontetrachloride-induced chronic hepatic injury and find possible mechanisms ofhepatic injury, pathologic model of chronic hepatic injury in mice caused bycarbon tetrachloride was set up by repeatly injecting of CCL4.Briefly,25%CCL4 mixture with olive oil subcutaneously twice per week at 2 ml?kg-1 bodyweight. Rats injected with olive oil alone served as controls. After 8 weeks ofthe experimental period , The experiment animals were treated withrhKD/APP at daily dose of 3, 6 ,12mg?kg-1 The control group received equalamounts of saline given intraperitoneally .After 28 days of the experimentalperiod,the rats were killed and blood was withdrawn from the abdominal aortafor the assay of serumenzyme(ALT,AST,TP,CHE,ALP,ALB,A/G,γ-GT,T-BIL,SA,Hyp)activities.Liver section were taken from each lobe of liver. rhKD/APP cansignificantly decresse the activities of ALT AST and the content of Hyp,andihhibit the decreasing of ALB ,A/G and hepatic fibrosis. rhKD/APP hasprotective effects on experimental liver injury in rats.The rhKD/APP was expressed in 80L fermentor with optimizingparameters and the broth was harvested with yield of 1.0 g·L-1,Firstly,weconstruct the methods of fermentation of rhKD/APP on large scale andpurifying of rhKD/APP. rhKD/APP have effects on prolonging the survival ofrat hepatocytes with effective dose at 2.5 μg·ml-1 and maximally effective doseat 10 μg·ml-1.on the pharmacology research ,the rhKD/APP was first foundthat it has ptotective effects on acute and chronic liver injury in rats.