Purification of recombinant growth hormone by clear native gels for conformational analyses: preservation of conformation and receptor binding.
Most protein preparations require purification steps prior to biophysical analysis assessing protein stability, secondary structure and degree of folding. It was, therefore, the aim of this study to develop a system to separate and purify a protein from a commercially available medicinal product, recombinant human growth hormone (rhGH) and show preservation of conformation and function following the gel-based procedure. The rhGH was run on clear native (CN) gels and recovered from the gels by electroelution using D-Tube Dialyzer Midi under rigorous cooling. Melting point studies indicated preservation of the structural integrity. This finding was confirmed by synchrotron radiation circular dichroism spectroscopy (SRCD) revealing an identical folding pattern for the sample before and after electrophoretic separation and purification. Synchrotron small-angle X-ray scattering (SAXS) indicated that the sample was folded and monomeric, both before and after separation and purification, and that its shape corresponded well to the known crystal structure of GH. Binding properties of rhGH to a receptor-model system before and after clear native electrophoresis were comparable. This analytical and preparative approach to purify and concentrate a protein preserving conformation and function may be helpful for many applications in analytical, protein and stereochemistry.
Chen
Wei-Qiang
WQ
Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
Salmazo
Anita
A
Myllykoski
Matti
M
Sjöblom
Björn
B
Bidlingmaier
Martin
M
Pollak
Arnold
A
Baumgärtel
Peter
P
Djinovic-Carugo
Kristina
K
Kursula
Petri
P
Lubec
Gert
G
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D013006:Growth Hormone