A hydroxymethylglutaryl-CoA reductase inhibitor synthesized by yeasts

Abstract Screening of different yeast species showed that they are able to synthesize hydroxymethylglutaryl-CoA (HMGCoA) reductase inhibitors. Crude methanol extracts and the purified inhibitors from Pichia labacensis and Candida cariosilignicola were tested for their biological activity on the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells. Identification of the inhibitors was studied by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.     

Decrease of proteolytic degradation of recombinant hirudin produced by Pichia pastoris by controlling the specific growth rate

The effects of the specific growth rate and methanol concentration on the degradation of hirudin produced by recombinant Pichia pastoris were investigated. When a methanol-limited state and the specific growth rate of 0.02 h^–1 were maintained during the fermentation, a minimum degradation of hirudin and a maximum specific hirudin production rate were achieved. By this strategy, the production of intact recombinant hirudin Hir65 reached 0.7 g l^–1 in fed-batch fermentation. Its proportion was 35% to all forms of hirudin.     

Methanol determination in Pichia pastoris cultures by flow injection analysis

An automated reverse flow injection analysis (r-FIA) system using stop-flow technique for quantifying methanol based on the enzymatic reactions of alcohol oxidase and peroxidase was developed. The system permitted methanol analysis in a linear range of 0.006-0.1 g methanol l^–1 without external dilution, and with a sampling frequency of 12 analyses per hour, with a relative standard deviation of 1.16%. The analyser was validated analysing samples from a Pichia pastoris fermentation producing a heterologous protein.     

Alcohol fermentation of enzymatic hydrolysate of exploded rice straw by Pichia stipitis

The xylose in an enzymatic hydrolysate of steam-exploded rice straw was not consumed by Pichia stipitis until the glucose was almost exhausted. A diauxic lag of 2 to 3 h in both cell growth and ethanol production occurred as metabolism switched from glucose to xylose utilization. Ethanol production was maximal [6 g ethano/l from 15 g reducing sugars/l (78% theoretical yield)] at an aeration rate of 0.2 vol/vol. min.The author was with the Department of Chemical Engineering, Kanazawa University, Kanazawa 920, Japan, but is now with the Engineering Biosciences Research Center, Cater-Mattil Hall, The Texas A&M University System, College Station, Texas 77843-2476, USA.     

植酸酶phyAm基因结构延伸突变改善酶的热稳定性

将来源于黑曲霉N25的植酸酶基因phyA^m重组于大肠杆菌表达载体pET-30b（＋）,以重组表达载体pET30b-FphyA^e为模板经PCR扩增获得结构延伸突变植酸酶基因phyA^m（在植酸酶基因C端增加了来源于pET-30b-FphyA^m载体上13氨基酸残基）。含突变基因的重组表达载体pPIC9k-phyA^e在GS115酵母中表达。纯化的突变酶pp-NP^e与野生型酶PP-NP^m-8相比：PP-NPA^e的最适反应温度上升了3气,75℃处理10min,热稳定性提高21％,比活力略有提高。最适反应pH为5．6,有效pH范围pH4,6到pH6．6。比未突变酶扩大了0.4单位。     

人血清白蛋白-睫状神经营养因子突变体融合蛋白基因在毕赤酵母中的表达及表达产物的纯化和活性鉴定

为了延长人睫状神经营养因子突变体的体内半衰期 ,将人血清白蛋白 (HAS)的C 末端和人睫状神经营养因子突变体AX15 (R13K)的N 末端通过一个 11个氨基酸的连接肽融合在一起 ,构建了融合蛋白HAS-AX15 (R13K)。HAS-AX15 (R13K)融合蛋白基因在巴斯德毕赤酵母中进行表达后通过阳离子交换层析、反向层析和凝胶过滤对表达产物进行了分离纯化。体外TF 1细胞存活实验表明与HAS融合并未影响AX15 (R13K)的生物学活性。体内动物实验表明HAS-AX15 (R13K)融合蛋白的疗效比AX15 (R13K)更为持久 :每 3天注射一次 4 8mg/kg的HAS-AX15 (R13K)融合蛋白的治疗效果优于每天注射一次 1 6mg/kg的AX15 (R13K)的治疗效果。HAS-AX15(R13K)融合蛋白不但可以减少用药次数 ,提高病人的顺应性 ,而且还可以减少用药量和血药浓度的波动 ,从而降低副反应 ,在临床应用上具有一定的优势。     

巴斯德毕赤酵母表达的突变型人白细胞介素-2的发酵条件与纯化研究

在以前的研究中,通过蛋白质工程技术获得了三突变体白细胞介素_2基因(编码125位半胱氨酸→丙氨酸;18位亮氨酸→蛋氨酸;19位亮氨酸→丝氨酸) ,并在毕赤酵母中加以表达。进一步优化表达条件,其最适诱导条件:80 %以上的通气,诱导2d ,初始pH60 ,甲醇终浓度为10%。在上述条件下表达量占菌体总蛋白的30%以上,大约200mg L。建立了一套从毕赤酵母表达上清中分离纯化分泌型表达蛋白IL_2的方法,经离心,超滤浓缩,强阳离子交换S柱和分子筛层析得到纯化的突变型和野生型IL-2 ;其得率为27% ,纯度达电泳纯并且HPLC检测只有一个峰。纯化的突变蛋白对CTLL-2细胞具有刺激性;与野生型IL-2相比,在各种温度条件下储存的突变蛋白保留有更高的活性;突变型IL-2的活力是野生型的4～5倍,具有更高的利用价值。     

猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ分泌型重组表达载体, 电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDS-PAGE和Western blot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,其表达量为108mg/L,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFN-γ基因的分泌表达。     

人Mn-SOD cDNA的克隆及其在巴斯德毕赤酵母中的表达

以人肝细胞株(L02)总RNA为模板,用RT-PCR扩增出人锰超氧化物歧化酶(hMn-SOD)cDNA,将其插入含有AOX1基因启动子和α分泌信号肽序列的毕赤酵母表达载体pPIC9k,构建重组质粒pPIC9k-MnSOD,转化毕赤酵母GS115,筛选出整合了多拷贝hMn-SOD基因的Mut^ 表型菌株,摇瓶培养,0．5％甲醇诱导表达。SDS-PAGE分析显示,诱导4d的培养上清中hMn-SOD的表达量约为上清总蛋白的32％,酶比活可达247、7u／mg。hMn-SOD在巴斯德毕赤酵母中实现了分泌性表达。