High hydrostatic pressure refolding of highly hydrophobic protein: a case study of recombinant human interferon β-1b from inclusion bodies

Purposes: Solubilization of inclusion bodies expressed in E. coli is a critical step during manufacturing of recombinant proteins expressed as inclusion bodies. So far, various methods have been used for solubilization and purification of inclusion body proteins to obtain active proteins with high purity and yield. The aim of this study was to examine the benefit of organic solvents such as alcohols in solubilization of recombinant interferon β-1b inclusion bodies. Methods: Effect of important parameters inclusion pH, concentration and type of denaturant and concentration of alcoholic solvents were optimized to formulate a suitable solubilization buffer and investigate their effect on solubilization of interferon β-1b inclusion bodies. Results: Our findings showed the acidic pH in the range of 2-3 is more suitable than alkaline pH >12 for solubilization and achieving higher content of interferon β-1beta and pure recombinant protein. We have also demonstrated that 1% SDS acts better than 2M urea to solubilize Inclusion body proteins of interferon β-1b at pH of 2-3. The interferon concentration was 2.35 mg per 100 mg IB when we used 40% (v/v) 1-propanol and 20% (v/v) 2-butanol into the buffer solution as well. Conclusion: The optimized method provides gentile condition for solubilization of inclusion body at high protein concentration and purity with a degree of retention of native secondary structure which makes this method valuable to be used in production and research area.

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APPLICATION OF HIGH HYDROSTATIC PRESSURE FOR RECOVERY OF BIOLOGICALLY ACTIVE CRUZAIN FROM RECOMBINANT INCLUSION BODIES

The FASEB Journal ◽

10.1096/fasebj.25.1_supplement.909.3 ◽

2011 ◽

Vol 25 (S1) ◽

Author(s):

Rosa Maria Chura Chambi ◽

Laura Simone Lemke ◽

Natália Malavasi Vallejo ◽

Luiz Juliano Neto ◽

Ligia Morganti

Keyword(s):

Hydrostatic Pressure ◽

High Hydrostatic Pressure ◽

Inclusion Bodies ◽

Biologically Active

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Survival of mouse blastocysts after low-temperature preservation under high pressure

Acta Veterinaria Hungarica ◽

10.1556/avet.52.2004.4.10 ◽

2004 ◽

Vol 52 (4) ◽

pp. 479-487 ◽

Cited By ~ 7

Author(s):

Cs. Pribenszky ◽

M. Molnár ◽

S. Cseh ◽

L. Solti

Keyword(s):

Phase Transition ◽

Hydrostatic Pressure ◽

Low Temperature ◽

High Hydrostatic Pressure ◽

Room Temperature ◽

Freezing Point ◽

Significant Loss ◽

Embryo Cryopreservation ◽

Subzero Temperatures ◽

Survival Capacity

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21°C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0°C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.

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