The effects of arginine on refolding of aggregated proteins: not facilitate refolding, but suppress aggregation

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Abstract

Arginine is one of the universal reagents that are effective in assisting refolding of recombinant proteins from inclusion bodies. The mechanism of the effects of arginine on refolding has remained, however, to be elucidated. Here we show that arginine does not stabilize proteins against heat treatment, as demonstrated by little change in melting temperature. It does increase reversibility of thermal melting and reduce aggregation under thermal stress. The observations suggest that arginine may not facilitate refolding, but may suppress aggregation of the proteins during refolding.

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Materials and methods

RNaseA and hen lysozyme were used for this study. RNaseA, and hen lysozyme, and BSA were dissolved in 40 mM Tris–HCl, pH 7.5. RNaseA and hen lysozyme were also dissolved in 1 M arginine or 50 mM glycine–NaOH, pH 10. RNaseA concentration was adjusted to 2.0 mg/ml for absorbance and to 1.7 mg/ml for circular dichroism (CD) measurements. Hen lysozyme concentration was adjusted to 0.4 mg/ml for absorbance measurements. Hen lysozyme concentration for CD measurements was 0.295 mg/ml. Protein concentrations

RNaseA

Thermal unfolding was monitored by a decrease in absorbance with time. Table 1 shows the mid-temperature, Tm, of the thermal unfolding in the absence or presence of 0.1–0.4 M arginine at a scan rate of 0.1 or 0.5 °C/min. In all cases above, the values of Tm are essentially independent of the scan rate, indicating that thermal unfolding of RNaseA is a reversible process and is in equilibrium under the conditions used. The reversibility was also confirmed by the second thermal scan, which was done

Discussion

We have shown here that arginine suppresses aggregation of two proteins, RNaseA, and hen lysozyme, different in chemical and physical properties. Suppression of aggregation was observed at a widely different arginine concentration from 0.1 to 2 M and at both neutral and high pHs. For hen lysozyme, arginine made the thermal unfolding reversible at neutral pH, while lysozyme developed turbidity without arginine. At both neutral and high pHs, addition of arginine to the protein did not lead to

Acknowledgements

This project is supported in part by the Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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Abbreviations: RNaseA, bovine ribonuclease A; CD, circular dichroism.

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