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| Efficient Soluble Expression and Oxidative Stability of Recombinant Mouse Ribonuclease Inhibitor |
| GE Ying, SUN Jing-hui, YAN Zheng, WANG Nan |
| Chinese Academy of Medical Sciences & Peking Union Medical College, Institute of Materia Medica, Beijing 100050, China |
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Abstract Ribonuclease inhibitor (RI) is a wide spread cytoplasmic protein regulating RNase activity in vivo, and extensively used in a variety of molecular biology applications where RNase contamination is a potential problem. Commercial mouse RI (mRI) was suggested to be more resistant to oxidative denaturation. In order to obtain the soluble active mRI product, recombinant expression vectors for mRI were constructed. Recombinant plasmid of pQE80L was more efficiently transformed into BL21 host cells than vector of pQE30, suggesting a toxic effect on the strains caused by basal expression of mRI. Upon induction with IPTG, mRI protein was expressed in both supernatants and inclusion bodies in different strains. BL21 strain yielded a similar amount of soluble products with higher RNase inhibiting activity than Top10 and TG1 strains. The yield of mRI fused to His-tag was about 4~8 mg/L. Expression of mRI fused to TrxA was also induced, which showed a partial inhibitory activity to RNase and restored to full activity after cleavage of TrxA from the protein. When mRI was treated with H2O2, the same oxidative stability was observed as to human RI (hRI), contradicting to the prediction that mRI should be more resistant to oxidative denaturation based on amino acid sequence analysis.
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Received: 07 June 2010
Published: 19 November 2010
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