| Summary: | 碩士 === 國立中央大學 === 化學研究所 === 100 === In recent years, the research combined protein function and structure analysis has become a famous area in the proteomics field. Through the protein crystallization method, high resolution of protein structure is able to be determined so as to provide important information relative to function and activity of enzyme. Currently, by the use of genetic deletion, insertion and point mutation methods to manipulate protein function is the major way to understand the relationship between protein function and structure in enzymology science. However, how to obtain a good quality of protein crystal is the most difficult issue in the protein structure determination.
Herein, our study is investigating the cytoplasmic region of human growth hormone receptor, and this part contains three important structural and functional sequence motifs. First one is proline-rich motif called Box 1, which is critical for association with JAK2. Second one is six tyrosine resides region playing important signaling transduction pathway through GH-induced phosphorylation; Last one is UbE (ubiquitin-dependent endocytosis) motif and it mediates GH-induced GHR ubiquitination.
Since the outer membrane region structure of growth hormone receptor had been reported, interactions between the outside structure of growth hormone receptor and hormone have been well known. In order to establish the signal transduction pathway of growth hormone receptor function from outer to inner membrane, therefore our experiment is focusing on structure determination of the cytoplasmic region of Growth receptor hormone through high quality of protein crystals and X-ray crystallography methods. According to protein structure information, the details of growth hormone induced signal transduction pathway in inner membrane are able to be completely understood. eg, GHR ubiquitinated mechanism, proliferation and and cell differentiation and diseases related to GHR disorder. .
Here we report that the cytoplasmic region peptide of Growth hormone receptor with over 95% purity by using Co2+ metal affinity resin interacting with six histidines on C-terminal of GHR and then applying to Gel filtration column filtering by different molecular weight. Interestingly, the result SDS-PAGE gel of gel filtration column shows that most growth hormone receptor forms dimer and tetramer complexes.
In addition, the result of crystallization is a good start to determine protein structure of the human growth hormone receptor cytoplasmic region, although crystal forms are not single crystals s. In the future, our studies will concentrate on finding better growth conditions for single protein crystallizations so that the determination of GHR protein structure can be achieved.
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