讨论：GST-融合表达
丁香园论坛
1811
我在两处看到GST-融合蛋白表达的方法有些困惑（主要集中在前几步）希望大家讨论。
1、 1）将带外源DNA的pGEX载体转化入感受态，LB/Amp生长12-15hr。
2）挑克隆于2mlLB/Amp中3-5hr
3）加入100mmol/L IPTG至终浓度0.1mmol/L诱导表达，继续3-5hr
4）。。。
5）。。。
2、1）On the first day, transform E. coli strain DH5α with the pGEX2TK–ER fusion expression vector and grow on LB agar plates with ampicillin. We found that many E. coli strains can be used to express GST-fusion proteins as long as the protein involved can be expressed at the desired level.
2）On the second day, pick a single colony into 2 ml of LB liquid medium with 100 μg ml−1 ampicillin and culture overnight at 37°C.
3）On the third day, transfer the 2 ml of bacterial culture into 1 l of LB liquid medium with 100 μg ml−1 ampicillin and culture overnight at 37°C.
4）On the fourth day, transfer the 1 l of bacterial culture into 4 l of LB liquid medium with 100 μg ml−1 ampicilline and cultured overnight at 37°C.
5）After growing for 2–3 h and once the optical density at 600 nm (OD600) reaches 0.8, the culture temperature should be lowered to 30°C and IPTG added into the culture to 100 μM final concentration.
6）Culture the bacteria for another 3 h and then collected by centrifugation at 5000 rpm for 10 min at 4°C.
第二中方法培养时间明显加长，与第二中方法有无区别？望指教
1、 1）将带外源DNA的pGEX载体转化入感受态，LB/Amp生长12-15hr。
2）挑克隆于2mlLB/Amp中3-5hr
3）加入100mmol/L IPTG至终浓度0.1mmol/L诱导表达，继续3-5hr
4）。。。
5）。。。
2、1）On the first day, transform E. coli strain DH5α with the pGEX2TK–ER fusion expression vector and grow on LB agar plates with ampicillin. We found that many E. coli strains can be used to express GST-fusion proteins as long as the protein involved can be expressed at the desired level.
2）On the second day, pick a single colony into 2 ml of LB liquid medium with 100 μg ml−1 ampicillin and culture overnight at 37°C.
3）On the third day, transfer the 2 ml of bacterial culture into 1 l of LB liquid medium with 100 μg ml−1 ampicillin and culture overnight at 37°C.
4）On the fourth day, transfer the 1 l of bacterial culture into 4 l of LB liquid medium with 100 μg ml−1 ampicilline and cultured overnight at 37°C.
5）After growing for 2–3 h and once the optical density at 600 nm (OD600) reaches 0.8, the culture temperature should be lowered to 30°C and IPTG added into the culture to 100 μM final concentration.
6）Culture the bacteria for another 3 h and then collected by centrifugation at 5000 rpm for 10 min at 4°C.
第二中方法培养时间明显加长，与第二中方法有无区别？望指教
