Runaway Replication Plasmid를 이용한$\gamma$Phage $cI_{857}$ Repressor 단백질의 생산 및 정제

Production and Purification of $\gamma$ Phage $cI_{857}$ Repressor Protein by the Use of a Runaway Replication Plasmid Vector

Runaway replciation plasmid pSY35AT를 이용하여 이 plasmid가 가지고 있는 $cI_{857}$ repressor를 숙주 MC1065를 이용하여 $30^{\circ}C$에서 $37^{\circ}C$로 온도를 올려 shift-up 법으로 생산하였다. $cI_{857}$은 wild type cI repressor 정제법을 수정하여 사용하였으며 정제된 $cI_{857}$ repressor 농도는 0.11mg/ml이었다. 그리고 정제된 $cI_{857}$ repressor와 $^3H-CTP$로 labelinf한 PrOr과의 결합활성은 cell 파쇄액보다 약 23배 높았으며, 온도가 올라갈수록 결합활성은 떨어졌다.

Runaway replication plasmid pSY35AT was used for the production of $cI_{857}$ repressor protein. E, coli MC1065 having plasmid pSY35AT was shifted from $30^{\circ}C$ to $37^{\circ}C$ $cI_{857}$ repressor protein produced was purified by a modification of the purification method of wild type cI repressor. The concentration of purified $cI_{857}$ repressor protein was 0.11 mg/ml. The binding assay of $cI_{857}$ repressor and right promoter - right operater ($P_RO_R$) labeled with $^3H-CTP$ was done. Relative activity of purified cIx5, repressor was higher about 23 times than that of cell free extract. A higher value of the temperature in the binding assay led to a lower value of the binding activity.