| Thymosinα1 (Tα1) is an acidic polypeptide composed of 28 amino acid residues. Itsrelative molecular mass is 3108Da and isoelectric point is 4.2. As a biological responsemodifier, Tα1 can significantly improve immune function. Thomas L and his research groupreported that the three fragments located in the position 17-24, 20-25 and 20-27 of Tα1 showedimmunological activities similar to and even better than the whole peptide. In recent years, Tα1has been widely used as immunoenhancer in clinical treatment of various immune deficiencydiseases, autoimmune diseases, tumors and virus infections. When used with other biologicalresponse modifier such as IL-2, IFN-αand thymic factor, a synergy effect can be achieved,especially for the treatment of chronic viral hepatitis. With the further confirmation of theexcellent effect of Tα1, its clinical applications extend significantly and its market demand isalso increasing. At present, most of thymosin in clinical is a variety of mixtures of peptidesextracted from animal tissues. The biological activities of preparations of thymosin fromdifferent producers have significant difference, and the products may be contaminated by theheterologous protein which may cause an allergic reaction when injected. The chemicalsynthesis process is too complicated which result in high cost of production and high price ofthe product. The serum half-life of Tα1 is too short, only two hours, which results in thefrequent administration to maintain its effective plasma concentration. Moreover, Tα1 isrequired for a long term use and high dosage in clinical which greatly hinders its wider use.In order to obtain high quality and good effect thymosin agents, researchers are exploringgenetic engineering methods for the preparation of thymosin. Until now, there are many reportsabout the expression of Tα1 in a variety of systems such as E. coli, yeast and plant. However,due to its small molecular weight which is only 3018Da, simply to express its monomer wouldcreate great difficulties to downstream separation and purification. Therefore most researchersconstruct Tα1 concatemer or fusion protein to increase its molecular weight to facilitate itspurification. However, according to the current reports, neither fusion expression norconcatemer expression has a high expression level. With the continuous expanding clinical application of Tα1, its market demand is increasing day by day. Therefore, the development ofhigh expression, high stability and longer half-life Tα1 agents are our pressing issues.Human serum albumin (HSA) is composed of 585 amino acids and its relative molecularweight is 68000Da. HSA is an inert protein of which structure is very stable. Its half-life is aslong as 19 days and has no significant enzyme and immune activity. HSA is an importantcarrier in blood. The spatial structure of HSA shows that C-terminus and N-terminus is on theopposite position which means that the target protein can be directly fused to the C-terminus orN-terminus. HSA is considered as the optimal carrier protein to improve the drug half-life.Human serum albumin fusion technology is to recombine HSA and medical protein's cDNAinto one plasmid, and then transfer it into the host cell for expression of the fusion protein. Atpresent, HSA has been expressed in a variety of systems such as E. coli, yeast, plants andtransgenic animals; However, HSA contains a large number of disulfide bonds and has a largemolecular weight, the expression level is significantly different in various systems. Pichiapastoris is the most successful one of these expression systems, and the level of secretedexpression of HSA in fermentor reached 10g/l. Therefore the majority of fusion expressionswith HSA are in Pichia pastoris. Currently some drugs that fused with HSA have entered intothe phase of clinical trials. Albuferon which is a fusion protein of HSA and interferon-αhascompleted its Phase III clinical trials in March 2009. The results showed that the half-life ofIFN-αafter fusion is as long as 18 times of the original half-life and the treatment efficacy andsafety are also satisfactory. Therefore, in this experiment we fused these three active fragmentsat the carboxyl-terminus of human serum albumin respectively and expressed these three fusionproteins in Pichia pastoris not only to achieve the purpose of the extension of its half-life, butalso to facilitate the downstream purification process.1. The construction of the expression vector pPICZαC-HSAWe extracted RNA from the human liver tissue and obtained the cDNA of HSA throughRT-PCR whose primers were designed as expression primers with Bspt104I and KpnIrestriction sites. 1% agarose gel electrophoresis showed that: A product of approximate 1800bpwas amplified. And then the purified product was digested with Bspt104I and KpnI and clonedinto the corresponding sites of vector pPICZαC. The recombinant vector pPICZαC-HSA wasconfirmed by the restriction enzyme XbalI and DNA sequencing. The sequencing data showed that there was no difference between the HSA sequence we cloned and the sequence reportedon NCBI which illustrates that we successfully constructed expression vector pPICZαC-HSA.2. The construction of the expression vectors pPICZαC-HSA-Tα117-24,pPICZαC-HSA -Tα120-25 and pPICZαC-HSA -Tα120-27The annealing primers were designed in accordance with the amino acid sequence in theposition of 17-24, 20-25 and 20-27 of Tα1 and codon preference of Pichia pastoris. Threeoligonucleotide fragments can be formed with the cohesive end of Eco81I (the restriction sitein the 3'terminus of HSA) and KpnI after annealing. In order to facilitate subsequent restrictionenzyme confirmation, AflII restriction enzyme site was designed. And then the three annealingpeptides of Tα1 carrying the cohesive ends of Ecor81I and KpnI were cloned into thecorresponding sites of vector pPICZαC-HSA. The transformants were digested with restrictionenzymes PstI and AflII and sequenced again. And the results demonstrated that three expressionvectors pPICZαC-HSA-Tα117-24, pPICZαC-HSA-Tα120-25 and pPICZαC-HSA -Tα120-27 wereconstructed successfully.3. The expression of fusion proteins HSA-Tα117-24,HSA-Tα120-25 and HSA-Tα120-27 in P.pastoris1) Screening of positive transformantsPlasmids were linearized with PmeI and were transformed into P. pastoris strain X33. Theinitial screening is through Zeocin-resistance. Then the transformants were selected throughgenomic PCR and the result showed that a product of 1800bp was amplified in some clones.The supernatants were harvested to run on the SDS-PAGE. The results indicated that theprotein, whose molecular weight was almost 64KD, was expressed which is in accordance withthe anticipated size. At the same time, the fusion protein was transfered onto a nitrocellulosemembrane for western blot. The membrane was incubated with rabbit anti-human Tα1polyclonal antibody (Bioss, China) followed by incubating with goat anti-rabbit IgGconjugated to HRP (Dingguo, China) and the fusion protein was visualized.2) To determine the desired induction time of the expression of HSA-Tα117-24,HSATα120-25 and HSA-Tα120-27 in P. pastorisCultivated the higher expression strain in BMGY / BMMY medium, sampled 0.5ml brothat 0h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, supernatants were run on SDS-PAGE for protein analysis. Electrophoresis results showed that the expression level increased with the prolongedinduction time and achieved a platform phase at the time of 120h. After that time point theexpression level remained unchanged, but the expression level of contaminate protein began toincrease. Therefore fermentation broth can be collected after the induction for 120h.3) To determine the pH value of the culture for the expression of HSA-Tα117-24,HSATα120-25 and HSA-Tα120-27 in P. pastorisCultivated the higher expression strain in BMGY and inoculated at 30°C until the cultureOD600 reached 2～6 and then every 10ml volume was transfered into centrifuge tubes(50ml).Cells were harvested by centrifugation and were respectively resuspended with 10ml BMMYmedium whose pH is 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0. After the induction of 120h, thesupernatants were run on the SDS-PAGE. The results showed that the optimal pH forexpression of fusion protein HSA-Tα117-24, HSA-Tα120-25 and HSA-Tα120-27 was 6.0.4) Preliminary study of purification process of fusion proteinsThrough the Mono Q HR5/5 anion column chromatography of ?KTA explorer 100, wedetermined that pH 10.0 is the optimum protein adsorption environment and the correspondingelution NaCl concentration is 0.3mol/L. After anion-exchange chromatography, we added0.1% TFA to the elution fractions containing the fusion protein and load the sample to thereversed-phase hydrophobic chromatography SourceTM30 RPC HR 10/30. The result showedthat 75% methanol (containing 0.1% TFA) can elute the protein sufficiently; methanol andTFA can be removed by rotary evaporation.To sum up: in this experiment, we constructed three fusion genes of HSA-Tα117-24, HSATα120-25 and HSA-Tα120-27 for the first time and successfully expressed them in Pichia pastoriseukaryotic expression system. At the same time we also completed the screening ofengineering yeast strain and optimized the desired induction time and pH of the culture for thesecreted expression of fusion protein in Pichia pastoris expression. Through ?KTA explorer100, we developed the primary purification process of fusion protein, as well as completed theinitial purification of the fusion protein through the anion-exchange chromatography andreversed-phase hydrophobic chromatography and laid the foundation for the development oflong half-life Tα1 agents.
|
PDF Full Text Request