cynR 조절 단백질은 T7 RNA polymerase/T7 promoter계에 의해 선택적으로 형질발현이 가능하였으며, 이로부터 DEAE-cellulose, phosphocellulose, 그리고 Sephadex G-200 컬럼 크로마토그래피를 이용함으로써 분리, 정제되었다. 분리된 cynR 조절 단백질은 그 분자량이 64,000 달톤으로, 두 개의 동일한 subunit으로 구성되어 있음이 확인되었고, 실제 N 말단의 아미노산 서열은 cynR 유전자의 염기서열로부터 예측한 것과 일치하였다. Gel retardation 실험을 통해 cynR 조절 단백질과 DNA의 특이적 결합을 조사한 결과, cynR 단백질은 cyn TSX operon과 cynR 유전자 사이의 짧은 intergenic region내의 영역에 특이적으로 결합하는 것이 밝혀졌다.
The cynR protein, which is a positive regulator for cynTSX operon in the presence of cyanate as well as a negative regulator for its own gene, was overexpressed by the T7 RNA polymerase/T7 promoter expression system in which the cynR gene was cloned under the control of T7 promoter. The cynR protein was purified on the serial columns of DEAE-cellulose, phosphocellulose, and Sephadex G-200. Throughout the purification, the cynR protein was identified as a single band of 32,000 daltons on SDS-polyacrylamide gel. The molecular weight of the protein in the native state was determined to be 64,000 daltons by HPLC, suggesting that the cynR protein exists as a dimer. The N-terminal acid sequence of the purified cynR protein was essentially the same as those deduced from the DNA sequence of cynR gene. In the gel retardation assay, the regulatory protein was specifically bound to the intergenic region between the cynTSX operon and the cynR gene. This result suggests that cynR protein binds to the intergenic region and regulates the expression of cynTSX structural genes and cynR regulatory gene simultaneously.
