| Background and PurposeHyaluronidases(HAase) were widely distributed in animal tissues and microorganisms. They were a group of endo-N-acetylhexosaminidases, which could degrade the glycosidic linkage between N-acetylglucosamine and D-glycuronic acid in hyaluronic aci(HA). The Mr of HA was reduced, so the viscosity and lubrication were decreased.HAase-mediated degradation of HA spurred the diffusion of the transudate from subcutaneous injections, local accumulation and blood, and it was beneficial for drug absorption. When combined administration with intravenous drip medicines, HAase could change the administration to subcutaneous injection or intramuscular injection, thus speeding up the absorption.Pichia pastoris(P. pastoris) has been extensively used as a host for expressing a variety of intracellular and extracellular proteins. It was widely employed for its many advantages, including fast culture, easy operating and industrial production. Importantly, it possessed the capacities of secretory expression, protein processing, protein folding, post translational modification and so on.Currently, the HAase which have been commercialized were extracted from animal tissues, such as cow and sheep. They have the shortcomings of low purity and high contents of foreign proteins. Cases have been reported that the patient's immune system was not tolerated with PH-20. In line with this, it appeared much desirable to develop strategies to produce PH-20 as a recombinant protein, which is superior to products that are routinely used in the clinics and conclusively could be of commercial relevance.In this paper, the eukaryotic expression of the ph-20 gene by P. pastoris heterologous gene expression system was studied.MethodsThe human ph-20 was coden optimized and total synthesized. A pair of primers with two enzyme sites Xho IⅨba I were designed. The ph-20 was inserted into the cloning plasmid pUC57. Using double digestion the nucleotide sequence of ph-20 was obtained from pUC57-ph20, and the gene fragment was inserted into P. pastoris secretory vector pPICZa A containing AOX1 promoter and a-secreting signal peptides, constructing a recombinant expression plasmid pPICZa A-ph20. The recombinant plasmid was translated into E. coli TOP 10 to amplify and then extracted. The positive plasmids were identified by enzyme digestioin, PCR and sequence analysis. After linearlized by Sac I, the recombinant plasmids were transformed into P. pastoris SMD1168H by electroporation, in which the plasmids would integrate into its genomes. The transformants were screened by Zeocin concentration gradients. We verified the phenotype of Mut+of the Zeocin-resistance transformants.The positive strains were cultured in flask first. After growing in YPD, BMGY and BMMY mediums, the cells were induced by 0.5%(v/v) methanol. The fermentation broths were centrifugated, and we assayed the activity of crude enzyme activity in the supernatant. The SDS-PAGE analysis showed that the Mr of the fusion protein was correct. To determine the optimal methods for assaying rhPH-20, we compared of three strategies:DNS, DMAB and turbidimetry methods, and the results were analyzed.The strain which had high expression level and high activity was used as engineered strain. It was induced and expressed in 1 L fermentor, and the fermentation conditions were optimized. Glycerol was selected as carbon source. When the cell wet weight was above 200 g/L and the glycerol was consumed, the methanol induction phase was started. Following centrifuged, the supernatants were ultrafiltrated by 5 kDa membrane, and the broths were then desalted and concentrated. Then the characteristics of rhPH-20 were investigated partly.Results and ConclusionThe length of ph-20 was 2009 bp, and its ORF is 1533 bp.511 amino acids were encoded, containing signal peptide sequence(1-35) and lyophobic anchoring zones484-511). Because the secretory ability of its signal peptides were not good and the proteins expressed in anchoring zone were undissolved, the two parts were got rid of. The remaining 448 amino acids sequences were optimized and synthesized. Its calculated Mr was 51 kDa. The recombinant expression plasmid pPICZa A-ph20 was constructed, and rhPH-20 was expressed successfully in P. pastoris for the first time.A Mut+phenotype of recombinant P. pastoris SMD1168H/pPICZa A-ph20 was screened under the Zeocin resistance concentration 4.5 mg/mL. Taking into account the posttranslation modification of P. pastoris expression system, the size of the rhPH-20 was approximately 58 kDa by SDS-PAGE analysis, which was larger than the calculated Mr.The linear ranges of DNS, DMAB and turbidimetry methods were 50-450 U/mL, 50-350 U/mL and 0-10 U/mL, respectively. The RSD were 2.71%,11.71% and 0.65%, respectively. DNS method was suitable for samples with higher activity, while turbidimetry was for the lower.In flask, the optimal expression conditions of this recombinant P. pastoris were determined as pre-induction A6oo=4, methanol concentration 0.5%, and induction time 72 h. The activity was up to 1×104 U/L. Then the optimization of fermentation was performed in 1 L fermentor. The optimal pre-induction cell wet weight and induction pH were 200 g/L and 6.0. The optimal flow rates of glycerol and methanol were 0.32 mL/(L-min) and 0.13 mL/(L-min), respectively. The dissolved oxygen was maintained above 20%. The highest rhPH-20 activity was 6×105 U/L after inducing 68 h. The total proteins were 0.307 g/L, and the specific activity was 1.954×103 U/L. The rhPH-20 reached up to 38% of the total proteins in medium supernatants.We also investigated the properties of rhPH-20:The optimum pH and temperature were found to be 6.5 and 37℃. It was relatively stable between5.5-6.5. Ca2+and Mg2+increased the activity when the ion concentration were 0.1-4 mmol/L. |
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