人DNA拓扑异构酶Ⅰ在毕赤酵母中的表达及发酵条件优化

Optimization of the Expression of Human DNA Topoisomerase Ⅰ in Pichia pastoris

为了在体外以人DNA拓扑异构酶Ⅰ (hTopoⅠ )为靶位进行抗肿瘤化合物的快速筛选 ,用RT PCR法从Hela细胞中克隆了hTopoⅠ基因ORF并在毕赤酵母中首次成功表达 ,表达产物可分泌到发酵上清 ,易于制备。蛋白酶A缺陷的重组酵母 (SMD hTopoI)分泌重组酶的能力比具有蛋白酶A活性的重组酵母 (X3 3 hTopoI和KM hTopoI)更高。通过发酵条件的优化 ,使用BMMY(pH 7 2 5) ,于 2 0℃ ,每隔 2 4h补加 0 5% (V V)的甲醇和 3 % (V V)的营养液 ,SMD hTopoⅠ诱导 72h后可表达最高的酶活力 (43 0 0 0u mL) ,发酵上清中hTopoⅠ可达 11mg L ,约占总蛋白的 10 %。SDS PAGE和Westernblot分析显示 ,表达的hTopoⅠ为 91kD蛋白 。

Human DNA Topoisomerase I (hTopo Ⅰ) has been identified to be an efficie nt targ et of many effective antitumor drugs. Natural hTopo Ⅰ is not convenient to be u sed in screening because of its low concentration in cells. In order to fast scr een new anticancer drugs targeting at hTopo Ⅰ from natural compounds i n vitro, hTopo Ⅰ gene open reading frame (ORF) has been successfully cloned and o verexpr essed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo Ⅰ specific antisense prim er and the hTopo Ⅰ ORF was synthesized by PCR. After digestion with EcoRⅠ and KpnⅠ, the synthesized fragment was inserted into pPICZαA, gave rise t o pPICZ α-hTopoI. After digestion with Sac I, the lined pPICZα-hTopoI was transforme d into Pichia pastoris strains(KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000ug/ mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD -hTopoI) could overexpress recombinant hTopo Ⅰ, which was fused to the α-fac tor secretion signal and could be secreted into the supernatant in the culture. α-factor could be cleaved from the expressed protein during secretion. A highe r activity amount of the enzyme was secreted by the particular strain SMD-hTop oI because of its absence of proteimase A than by other strains which possess p roteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-h TopoI was induced in BMMY (pH7 25) at 20℃, with addition of 0 5% (V/V) me thanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 4 3 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protei n in the culture supernatant. SDS-PAGE and Western blot analyses showed that th e mass of the recombinant hTopo Ⅰ was 91kD with no glycosylation.