研究了pH和温度对重组毕赤酵母表达retep lase(rPA)发酵过程中的降解现象。在低pH(4.5)诱导条件下,由于蛋白酶活性高,rPA降解严重,表达量几乎为0,提高诱导pH至6.5,蛋白酶活性降低,rPA的表达量最高达到122 m g/L。通过将诱导温度从30...研究了pH和温度对重组毕赤酵母表达retep lase(rPA)发酵过程中的降解现象。在低pH(4.5)诱导条件下,由于蛋白酶活性高,rPA降解严重,表达量几乎为0,提高诱导pH至6.5,蛋白酶活性降低,rPA的表达量最高达到122 m g/L。通过将诱导温度从30°C降低到20°C,rPA的表达量从153.2 m g/L提高到207.9 m g/L。降低温度能减少细胞死亡率,从而减少了发酵液中蛋白酶的释放,降低了蛋白酶的活性,抑制了对目的蛋白rPA的降解。另外,低温使胞内AOX酶活性提高,从而增强了rPA的表达。展开更多
The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the rec...The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been inte-grated into the genome of gametophytes of L. japonica, and the expression product showed the ex-pected bioactivity, implying the proper post-transcript modification in haploid gametophyte.展开更多
文摘研究了pH和温度对重组毕赤酵母表达retep lase(rPA)发酵过程中的降解现象。在低pH(4.5)诱导条件下,由于蛋白酶活性高,rPA降解严重,表达量几乎为0,提高诱导pH至6.5,蛋白酶活性降低,rPA的表达量最高达到122 m g/L。通过将诱导温度从30°C降低到20°C,rPA的表达量从153.2 m g/L提高到207.9 m g/L。降低温度能减少细胞死亡率,从而减少了发酵液中蛋白酶的释放,降低了蛋白酶的活性,抑制了对目的蛋白rPA的降解。另外,低温使胞内AOX酶活性提高,从而增强了rPA的表达。
基金Supported by the National High Technology Research and Development Program of China (Grant Nos. 20060110A4013 and 2006AA100311)National Natural Science Foundation of China (Grant No. 40506030)Innovative Key Project of the Chinese Academy of Sciences (Grant No. KZCX2-YW-209)
文摘The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been inte-grated into the genome of gametophytes of L. japonica, and the expression product showed the ex-pected bioactivity, implying the proper post-transcript modification in haploid gametophyte.
