Research On The Expression Of Recombinant Human Prethrombin-2 And Butyrylcholinesterase By Glycoengineered Pastoris

With the rapid development of genetic engineering, the demand for protein drugs is increased. All kinds of protein drugs expression platform appeared one after another, Pichia pastoris as one of the simplest eukaryotic expression system is more and more gets the attention of the people. But the wild yeast secretory glycoprotein existed high-mannose structure, witch limited the development of it as a glycoprotein generator. Early in our lab, using two homologous recombination technology successly knocked out Pichia pastoris mannose transferase genes and blocked high mannosylation of yeast glycosylation pathways. The gene of synthetic complicated mammal glycoform was integrated into the genomes of genetically engineered Pichia pastoris. The glycoengineered Pichia pastoris achieveed the humanized reformation and successfully used in the expression of protein drugs.a-Thrombin is involved in an important part of the protease in the process of blood coagulation. Butyrylcholinesterase (BChe) is widely distributed in the plasma, liver and lung and its the most main function is to hydrolysis butyrylcholine in the synapses, preventing neural signal transmission. There are most of commercial a-thrombin and BChe that are extracted from human plasma and mammal plasma. But the productions is restricted by the environment and the blood and are difficult to meet clinical needs.Glycoengineered yeast has quick and efficient expression of protein and carbohydrate modification ability of mammalian cells. This study based on glycoengineered yeast expression platform to express recombinant human prethrombin-2 and butyrylcholinesterase and explore application prospect of yeast in expressing heterologous glycoproteins by glycoengineered yeast.1. The study of prethrombin-2 expressed by glycoengineered yeastSynthesised prethrombin-2 gene was build in pichia expression vector, electrotransformed and screened reengineering yeast, which was induced by methanol. The culture supernatant was purified by SPFF and Unisp cationic chromatography purification, then purified 37 kda aimed protein.Through PNGase F digested purified sample, the molecular of aimed protein without carbohydrate chain became 35 kda, which was consistent with the theoretical size. Peptide mapping analysis results show that the purified glycoprotein of prethrombin-2.2. The study of Ecarin expressed by yeast and HEK 293T cellIn vitro, prethrombin activator Ecarin can activate prethrombin to thrombin. In order to get thrombin, we used pichia and 293T cells to express Ecarin. Ecarin sequence was fully genetically synthesized and built in pPICZaA and pcDNA(3.1).In pichia expression system, try to expressed intracellular forms and secreted form, but neither obtained the active Ecarin, Western Blot couldn’t detect specific band.Transfacted Ecarin into HEK 293T cells and collected the culture supernatant. Prethrombin-2 possessed by the culture supernatant of 293T/Ecarin cells,could digest S-2238 (H-D-Phe-Pip-Arg-pNA-2HCL) to produce yellow pNA. Prethrombin-2 possessed by the culture supernatant of 293T cells didn’t change color. It howed that HEK 293T cells had expressed Ecarin which could activate prethrombin-2 to thrombin.3. The study on activity of prethrombin-2Use chromogenic substrate absorbance values of color change Ecarin activation measuring prethrombin 2. When the amount of purified prethrombin 2 was constant, the increase of 293T/Ecarin culture supernatant, the absorbance of S-2238 degradation also increase, which meaned activation of prethrombin-2 by Ecarin has a dose dependent. Purificated prethrombin-2 of Ecarin activation, can promote the mice plasma agglutination, whose time was 16s. The blood clotting time of Thrombin as the positive control in TT kit was 27s. Those illustrated that prethrombin-2 had blood clotting activity after activation.4. The different forms of butyrylcholinesterase expressed by wild yeastThe aim of this experiment used glycoengineered yeast to prepare human recombinant butyrylcholinesterase. Express different structure of butyrylcholines-terase in the wild type yeast X33, compared expression quantity and activity of different structure butyrylcholinesterase. Then select long-acting butyrylcholines- terase as target protein ueed for glycoengineering yeast expression and enzyme activity research.Full gene synthesis butyrylcholinesterase sequence, was build into pichia expression vector, and transformed into the wild type yeast X33, then induced cultivated, culture supernatant was analysised by SDS-PAGE electrophoresis. There was no purpose protein bands in electrophoresis figure. There were color reaction by the expression of BChe culture supernatant digested substrate S-Butyrylthiocholine iodide (BSCh), after added the chromogenic agent DTNB through measured the absorbance value change. Comparing with 1/40 mice serum volume, BChe culture supernatant only was 80% of 1/40 mice serum enzyme activity, that expression amount was low.Using wild type yeast X33 try to express butyrylcholinesterase monomer.Natural butyrylcholinesterase is autotetraploid polymers,340 kda. Considering the relative molecular weight is large, it’s difficult for yeast to secret, thus built butyryl-cholinesterase monomer expression vector. According to the four polymers bound by butyrylcholinesterase C-terminal 40 amino acids, used PCR technology to build truncate butyrylcholinesterase (BCheC). BCheC was construct to the pPICZαA, the wild type yeast X33 express BCheC culture supernatant by SDS-PAGE electrophoresis analysis, there was no target protein electrophoresis figure that could be observed. By cholinesterase colorimetric analyzing method, there was butyrylcholinesterase activity in culture supernatant. But compared with the mice serum which diluted 40 times, its activity was low.Butyrylcholinesterase monomer half-life is far shorter than the four polymers. In order to extend its half-life, the truncated type butyrylcholinesterase (BcheB) fused with HSA and series expression. There was no target protein electrophoresis figure that could be observed. The culture supernatant have butyrylcholinesterase activity proved by cholinesterase colorimetric method, but compared with the mice serum dilution 40 times, its activity was low.Because tetramer structure of butyrylcholinesterase is the most stable. By adding of proline polymer (poly-proline, polyP) in the medium, it can promote monomer and dimer form four polymers in the culture. Considering the polyP molecular is small, easy to be degraded. Use the polyP fusion HSA and BChe coexpress scheme. Will gather polyP-HSA and butyrylcholinesterase genes into the same expression vector, transformed into wild yeast X33, then reengineered X33 induced by methanol. There was no target protein electrophoresis figure that could be observed. By cholinesterase colorimetric analyzing method, there was butyrylcholinesterase activity in culture supernatant. But compared with the mice serum which diluted 40 times, its activity was low.5. BCheB-HSA expressed and purified in glycoengineered yeastDue to BCheB-HSA protein molecular weight less than tetramer butyryl-cholinesterase protein molecular weight, longer half-life than monomer butyryl cholinesterase (BCheC). And through the expression of wild yeast X33 results indicate that BCheB-HSA expression quantity and enzyme activity and at the same level, so choose BCheB-HSA expressed and purified in a glycoengineering yeas. Through two step cation chromatography purification, SDS-PAGE bands could be observed in purpose and Western Blot showed that purpose of purified protein, but the purpose protein degradation had occurred. Enzyme cholinesterase colorimetry test showed that the sample of the purified enzyme activity of about 1/8 of the mice serum. As the sample dilution, absorbance values also fertilizations, subsequently showed obvious dose-dependent. X33 expressed BCheB-HSA culture supernatant after the same two step cation chromatography purification, was not prepared proved by Western Blot anti-HSA.This experiment based on glycoengineered yeast expression of exogenous glycoprotein-prethrombin-2 and butyrylcholinesterase, purified and analysised enzyme activity, proved glycoengineered yeast can expressed exogenous glycoprotein with the activity.