[Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris].

Qi-Wen Yu, Ning-Li Li, Hong Nie, An-Lun Ma, Bo Xi, Yi Gong, Dong-Qing Zhang
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Abstract

To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.

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[酵母毕赤酵母中表达的重组人ifn - β的纯化和鉴定]。
为了寻找一种高效、快速的纯化和鉴定酵母毕赤酵母中表达的重组人ifn - β (rhifn - β)的方法,本文比较了Blue Sepharose 6快速流法(Blue S6FF)和免疫亲和层析法(IAC)。rrhin - β在15升生物反应器中产生,并采用上述两种方法纯化。采用酶联免疫吸附法测定rrhin - β蛋白浓度和纯化rrhin - β蛋白中残留的小鼠IgG。通过PAGE和Western blot检测其分子量和特异性。通过凝胶扫描确定了特定沉淀带的密度。采用细胞致病效应抑制法(CPEI)测定其相对生物活性。结果表明,用Blue S6FF和IAC纯化2 l发酵上清液,分别可获得2.65和3.03 mg的rrhin - β。分子量为22 kD。rrhin - β的特殊沉淀浓度分别为95.1%和96.2%。Blue S6FF和IAC纯化的rrhin - β的相对生物活性分别为1.63 × 10(7) IU/mg和1.43 × 10(7) IU/mg。IAC纯化的rhifn - β中小鼠IgG残留量小于50 μ g/L。结果表明,采用IAC法可以有效、快速地从酵母毕赤酵母发酵上清液中纯化rrhin - β。Blue S6FF和IAC纯化的rrhin - β产物纯度和生物活性几乎相同。实验积累的数据对大规模制备rrhin - β有一定的参考价值。
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