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Abstract
Objectives: The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein.
Methods: Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms.
Results: Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an α-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained.
Conclusions: The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.
| Abbreviations | ||
| GH | = | human growth hormone |
| T2DM | = | type 2 diabetes mellitus |
| SPS | = | solid phase synthesis |
| CD | = | circular dichroism |
| SPR | = | surface plasmon resonance |
| TAT | = | 1,3,5-triacryloyl-hexahydro-s-triazine |
| TFE | = | 2,2,2-trifluoroethanol |
| MALDI-TOF | = | matrix-assisted laser desorption ionisation-time of flight |
| Wang resin | = | 4-(hydroxymethyl) phenoxymethyl |
| HBTU | = | 2-1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate |
| HOBt | = | N-hydrozybenzotriazole |
| HBS-EP | = | 10 mM HEPES, 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid (EDTA) and 0.005% Tween20, pH 7.4) |
| DMF | = | 20% in N,N-dimetilformamide |
| DIEA | = | N,N-diisopropylethylamine |
| TFA | = | trifluoroacetic acid |
| HPLC | = | high-performance liquid chromatography |
| ELISA | = | enzyme-linked immunosorbent assay |
| BSA | = | bovine serum albumin |
| E/S | = | enzyme–substrate ratio |
| SDS-PAGE | = | sodium dodecyl sulfate polyacrylamide gel electrophoresis |
| RU | = | resonance units |
| Abbreviations | ||
| GH | = | human growth hormone |
| T2DM | = | type 2 diabetes mellitus |
| SPS | = | solid phase synthesis |
| CD | = | circular dichroism |
| SPR | = | surface plasmon resonance |
| TAT | = | 1,3,5-triacryloyl-hexahydro-s-triazine |
| TFE | = | 2,2,2-trifluoroethanol |
| MALDI-TOF | = | matrix-assisted laser desorption ionisation-time of flight |
| Wang resin | = | 4-(hydroxymethyl) phenoxymethyl |
| HBTU | = | 2-1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate |
| HOBt | = | N-hydrozybenzotriazole |
| HBS-EP | = | 10 mM HEPES, 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid (EDTA) and 0.005% Tween20, pH 7.4) |
| DMF | = | 20% in N,N-dimetilformamide |
| DIEA | = | N,N-diisopropylethylamine |
| TFA | = | trifluoroacetic acid |
| HPLC | = | high-performance liquid chromatography |
| ELISA | = | enzyme-linked immunosorbent assay |
| BSA | = | bovine serum albumin |
| E/S | = | enzyme–substrate ratio |
| SDS-PAGE | = | sodium dodecyl sulfate polyacrylamide gel electrophoresis |
| RU | = | resonance units |