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Studies On The Recombinant Bovine Pancreatic Trypsin Inhibitor

Posted on:2007-09-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:L L YangFull Text:PDF
GTID:1100360185454762Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Bovine pancreatic trypsin inhibitor (BPTI,aprotinin for trade name) is apolypeptide of 58 amino acids with a molecular weight of 6510 dalton. BPTI is anatural non-specific serine protease inhibitor which can inhibit trypsin,chymotrypsin, kallikrein and plasmin etc.These years aprotinin is not limited to therapy of acute necrosispancreatitis(ANP), it has been widely used in arresting bleeding of blood duringcardio pulmonary bypass(CPB) cordis surgery, shock, asthma and gynecologicalillness etc. Aprotinin has become one of the important biochemicalpharmaceuticals for its protection for ischemic reperfusion injury of variousexperimental tissues such as liver, brain, and heart etc.However, the current aprotinin for commercial use was extracted frombovine organs such as lungs, pancreas etc. The complex manufacturing processbeyond to control led to impure components which were the major factors ofsevere clinical adverse effects. The source of starting material was limitedbecause of mad cow disease.Therefore, the clinical application of aprotinin wasconfined. On the other hand, with the continuous discovery for aprotininpharmacodynamics, increasing clinical application coverage and fast growingmarket demand, the only way to solve these problems will be the use of the geneengineering technology to produce recombinant BPTI(rBPTI).rBPTI has been reported to be expressed in E.coli, Saccharomyces cerevisiaeand Pichia pastoris, but there were some insurmountable flaws such as lowproductivity, incorrect folding and cleavage. Especially in Pichia pastoris, it wasfound that the Kex2 protease was not able to cleave off rBPTI to produce a correctN-terminal sequence "Arg-Pro-Asp" with signal peptide of alpha mating factor.Therefore, the expression system of rBPTI construction which structure iscompletely consistent with the natural BPIT(correct N-terminal and spatialconformation)and is stable, high-yield and easy to industrialized remains anunsolved problem for the production of rBPTI.On the basis of our original research on Pichia pastoris and human serumalbumin, we studied the production of rBPTI from the aspects of signal peptide,promoter, yeast strain and yeast preferred codons etc. At last, alpha mating factorsignal peptide was replaced by plasmid pPICZα with human serum albumin (HSA)signal peptide and Pichia pastoris expression systems was established that wasapplicable to industrialization. Additionally, rBPTI we produced was proved to bea secretory protein with a complete-modified natural structure and biologicalactivity. This research work included: ①Construction of vector pPICZ/hsasp-bpti,,transforming it into Pichia pastoris via electroporation and screening the highlevel strain. ② Purification of rBPTI and identification of the structure andbioactivity of rBPTI. ③Establishing a large-scale fermentation process of rBPTIin 80L fermentor and creating a new method to purify rBPTI. ④ Establishing thechronic liver injury model induced by carbon tetrachloride and observing theprotective effects of rBPTI on experimental chronic liver injury induced bycarbon tetrachloride in rats in vivo. ⑤ Observation of therapeutic effects of rBPTIon acute necrosis pancreatitis in rats in vivo.1. Construction of Pichia pastoris expression system for rBPTI andcharacterization(1)Construction of rBPTI expression vector pPICZ/hsasp-bpti: Twooligonucleotides of hsasp were synthesized which had restriction endonucleasecohesive ends of BstBⅠ and XhoⅠ , and after two oligonucleotides annealingDNA fragment was ligated into vector pPICZα which was digested withBstBⅠ and XhoⅠ. The plasmid was named pPICZ/hsasp. bpti gene was obtainedusing specific cloning primers by PCR, then it was inserted into T vector. Afteridentification by endonuclease digestion assay, Xho I and Xba I target sites wereadded using expression primers by PCR. After identified by endonucleasedigestion assay and sequencing, the expression vector pPICZ/hsasp-bpti waslinearized and transformed into Pichia pastoris via electroporation.(2)Screening of the transformed Pichia pastoris: Transformed yeasts wereplated on YPD plates containing Zeocin to isolate Zeocin-resistant clones. Afterclones were cultured for 72 hours at 28℃ , 20 single clones were picked up. Thenthe genomic DNA of the transformed yeasts was extracted to perform PCR usingthe expression primers. Yeast clones tested positively by PCR were proliferatedand then rBPTI was induced with 0.5% methanol. The supernatant offermentation was identified by SDS-PAGE, inhibition activity to trypsin and highlevel strain BPTIS7 was screened.(3)Expression and identification of rBPTI: BPTIS7 was proliferated in 2Lculture at temperature 28℃ and rBPTI supernatant was purified with Mono Scation exchange chromatography and SourceTM30 RPC hydrophobicchromatography. Results showed that there was single band in SDS-PAGE andpurity was more than 95%. MS showed that molecule weight was 6508D andtheory value was 6511D. Amino acid sequencing suggested that the 15th Nterminal amino acid was identical with natural BPTI.2. Studies on large-scale fermentation and purification process of rBPTI(1)Studies on large-scale fermentation process of rBPTI: Pichia pastoris is amonocellular eukaryon which has many advantages as an expression host, and it'ssuitable for large-scale expression of the extraneous proteins. With theidentification of the bioactivities of the rBPTI, the large-scale fermentationprocess of rBPTI was explored. Studies were focused on pH value, culturemedium, resolved oxygen, methanol feed speed, initial biomass etc. The bestconditions for fermentation were found as follows: FM21 medium (including0.5% peptone), yeast biomass 220g·L-1, pH 3.3, DO between 25~30% and thesupply speed of methanol 8.5~9.0ml/h/L initial fermentation volume. Theconcentration of rBPTI in the broth could be reached 200mg·L-1.(2) A new method to purify rBPTI at large-scale: The fermentationsupernatant was purified with SP Sepharose XL cation exchange chromatographyand SourceTM 30 RPC hydrophobic chromatography, then the eluate was distilledto condense rBPTI. The purity could be more than 95% and the yield coefficientwas higher than 60%.3. Protective effects of rBPTI on chronic liver injuryThis study was carried out to observe the protective effect of rBPTI oncarbon tetrachloride-induced chronic liver injury in rats. The chronic injuryfibrosis mice models were built using 40% carbon tetrachloride. rBPTI was givenat doses of small, medium and large and compared with hepatocytegrowth-promoting factors(HGF) which were used as controls . The levels of ALT,AST, TP, CHE, ALP, ALB, A/G, γ-GT, T-BIL,SA, Hyp were measured and thechange of hepatic histology was observed. Results showed that small, mediumand large doses of rBPTI compared with the control group decreased the values ofALT, AST,γ-GT, Hyp,T-BIL significantly (P < 0.01,P < 0.05) and increased thevalues of ALB,A/G,CHE significantly (P < 0.01,P < 0.05). There were nosignificant statistical difference of the changes of SA, ALP. The pathologicalchange of the small, medium and large doses group compared with the modelcontrol group were as follows: the structure of the hepatic phyllode was blurred,some liver cells were disintegrated and necrotic, there were moderately fatdenaturation in the central section of the hepatic sub-phylllode and some meetingsection, in the liver cells there were many visible different sized around vacuoles,the inflammation cells infiltrating and liver fibrosis were alleviated.4. Studies on therapeutic effects on rBPTI on experimental acute necrosispancreatitisThis study was to observe the therapeutic effect of rBPTI on acute necrosispancreatitis in rats. Acute necrosis pancreatitis was induced by injections of 3.5%sodium taurocholate into the main pancreatic duct of rats. Amylase and lipaseactivity were assayed and pathological changes were observed after treatmentwith rBPTI. Results showed that amylase and lipase were increased in modelsgroup. rBPTI markedly inhibited amylase, lipase activity and amelioratedpathological changes of acute necrosis pancreatitis. It indicated that rBPTI couldsuppress pathogenesis of acute necrosis pancreatitis in rats.To sum up, we expressed the rBPTI with correct N-terminal sequence inPichia pastoris with human serum albumin signal peptide;It was confirmed thatthe physiochemical character and bioactivity of rBPTI were identical with thenatural BPTI;we also built a new method for large-scale fermentation andpurification. We confirmed the protective effects of rBPTI on carbon tetrachloride-induced chronic liver injury in rats and the therapeutic effect of rBPTI on acutenecrosis pancreatitis in rats as well as natural BPTI. The current studies couldbuild theoretically and experimental foundation for research of rBPTI and thevalue of industry production were initially explored.
Keywords/Search Tags:recombinant bovine pancreatic trypsin inhibitor, human serum albumin signal peptide, Pichia pastoris, chronic liver injury, acute necrosis pancreatitis
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