Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
Yeast. 2010 May;27(5):285-92. doi: 10.1002/yea.1753.
The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris. The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A-base overhang. The other vector pYEVB, with the same features for blunt-end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes BfuI and SchI, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors, followed by selection on plates supplemented with X-gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lacO reconstruction strategy that can produce a full-length lacO to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by SalI and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time-consuming conventional cloning procedures.
酵母载体 pYEV 和 pYEVB 是为了在毕赤酵母中平行聚合酶链反应（PCR）克隆开放阅读框（ORF）和立即表达蛋白质而设计的。pYEV 载体用于克隆通过 Taq 或类似聚合酶混合物获得的 PCR 片段，这些酶会留下一个 A 碱基突出。另一个载体 pYEVB 具有用于 PCR 产物平头末端连接的相同特征，是为了与 pYEV 互补而开发的。这两个质粒分别使用 BfuI 和 SchI 限制酶线性化。纯化的 PCR 产物无需任何其他处理，即可克隆到线性化载体中，然后在添加 X-gal 的平板上进行选择。只有携带目标基因且方向正确的期望重组体才能产生典型的蓝色菌落。这种筛选技术基于 lacO 重建策略，可以产生全长 lacO 以耗尽内源性 LacI 并激活宿主中 lacZ 的转录。从蓝色菌落中提取的重组质粒可以通过 SalI 线性化，并转化为毕赤酵母进行立即表达。使用这两个载体，研究人员可以避免繁琐且耗时的传统克隆程序。
