High pressure refolding of recombinant human growth hormone from insoluble aggregates. Structural transformations, kinetic barriers, and energetics.
Two different types of insoluble, non-native aggregates of recombinant human growth hormone were formed by agitation in buffer or buffer containing 0.75 m guanidine HCl (GdnHCl) and characterized by infrared and second derivative UV spectroscopies. The degree of secondary structural perturbation was greater in the aggregates formed in 0.75 m GdnHCl. Both aggregate types were dissolved and refolded using high hydrostatic pressures in combination with either elevated temperature or non-denaturing levels of guanidine HCl or urea. The effects of a range of temperature, pressure, and chaotrope concentrations were tested and led to optimal conditions that approached 100% yield of native protein. The aggregates formed in 0.75 m GdnHCl required higher concentrations of urea or GdnHCl, or higher temperature or pressure for a yield equivalent to that for aggregates formed in buffer alone. Investigation of the effects of pressure, temperature, and chaotrope on unfolding of rhGH documented that under conditions used for optimal high pressure disaggregation and refolding, the native state is greatly favored thermodynamically (e.g. 25 kJ/mol at 2000 bar and 0.75 m GdnHCl). Dissolution of aggregates under pressure is a kinetically limited process. Comparison of refolding yields in GdnHCl and urea solutions suggest that pressure effects on electrostatic interactions do not dominate pressure effects on disaggregation. We suggest that non-native hydrogen bonds between protein molecules within aggregates of recombinant human growth hormone are responsible for the rate-limiting kinetic barrier in pressure-induced disaggregation.
St  John
R J
RJ
Department of Chemical Engineering, Engineering Center ECCH 111, University of Colorado, Boulder, CO 80309, USA.
Carpenter
J F
JF
Balny
C
C
Randolph
T W
TW
D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D019382:Human Growth Hormone / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011312:Pressure; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D019382:Human Growth Hormone; D014508:Urea; D019791:Guanidine