摘要
为探讨亮氨酸拉链蛋白与DNA的结合机理,设计了含有GCN4亮氨酸拉链蛋白基区结合DNA的必需的35个氨基酸的折叠片段,并将其克隆到Esche^chia coli BL21表达。以ZG1(+)、ZG1(-)、ZG2(+)、ZG2(-)、ZG3(+)、ZG3(-)寡聚核苷酸片段在PCR自动热循环系统连接得到亮氨酸拉链蛋白基因片段,将此片段克隆到pET3b质粒中,酶切后用2%琼脂糖电泳检测证明克隆成功,将这种重组质粒转化到E.coli DH5α,发现pET3b重组体在E.coliDH5α中表达成功,从转化子中分离得到重组质粒pET3b,序列分析证明插入序列为合成的亮氨酸拉链蛋白基因;将重组质粒pET3b在E.coli BL21于5mL含有50 μg·mL^(-1)氨苄青霉素和34 μg·mL^(-1)氯霉素的LB液体培养基中以IPTG为诱导剂,37℃下表达,10%SDS-PAGE检测到外源基因蛋白质分子量为4000 Da左右;重组体在10 L含氨苄青霉素和氯霉素的LB液体培养基中、28℃时能大量表达。
To detect the mechanism of bZIP binding DNA, we designed a small folding domain containing the 35 kinds of amino acids necessary for DNA binding of the basic leucine zipper protein GCN4 and cloned it into Escherichia culi BL21 to express. Oligonucleotides ZG1 ( + ) 、 ZG1 (- )、 ZG2 ( + ) 、ZG2 ( - )、 ZG3 ( + )、 ZG3(-)were used to synthesize the leucine-zipper protein domain by Authorized Thermal Cyster for PCR. The synthesized domain was cloned to plasmid pET3b and then transformed into E. culi DH5 α. It could expressed successfully in E. culi DH5 α. It was identified that inserted domain was the synthesized genes by restriction enzyme analysis and sequencing. The recombinant Escherichia coli BL21 was incubated in 5 mL LB liquid medi- um including 50 ug · mL^(-1) ampicillin and 34 ug · mL^(-1) chloramphenicol till it get to exponential phase. In- duced the culture by IPTG at 37℃ to express in small scale. The induced solution was tested with 10%SDS- PAGE and showed the expressed protein of inserted DNA domain was about 4000 Da. The inserted domain could express induced by IPTG at 28℃ in large scale.
出处
《化学与生物工程》
CAS
2003年第5期35-37,共3页
Chemistry & Bioengineering
基金
国家留学基金委资助(99842135)
