EXPRESSION AND PURIFICATION OF RECOMBINANT PROINSULIN ASPART ASPS.RWR ON PICHIA PASTORIS X33
Type 1 Diabetes is a disease that characterized by high level of blood glucose due to low production of insulin inside the body. Main treatment for type 1 diabetes is through regular insulin injection for the patient. To shorten the time for insulin injection to take effect, the amino acid sequences...
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| Format: | Final Project |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/67511 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Type 1 Diabetes is a disease that characterized by high level of blood glucose due to low production of insulin inside the body. Main treatment for type 1 diabetes is through regular insulin injection for the patient. To shorten the time for insulin injection to take effect, the amino acid sequences of the insulin is modified. Insulin that is modified through this way is called insulin analog. One of the insulin analogs that is already marketed widely is insulin aspart, which has a change in its B28 residue from proline to aspartic acid. One of the known host microorganism for insulin production is yeast Pichia pastoris. Yeast P. pastoris can utilizes methanol as inducer to express the desired protein. One of the P. pastoris strains, namely P. pastoris X33 can alo uses methanol as its sole carbon source. In this study, recombinant proinsulin aspart AspS.RWR is expressed in yeast Pichia pastoris X33. The expression is done in two media; Buffered Methanol-complex Medium (BMMY) and Basal Salt Medium (BSM). The AspS.RWR is then purified with cation-exchange chromatography. The expression of AspS.RWR is done under condition: pH 6,00 (BMMY)/5,00 (BSM); starting OD600 of 60; aeration ratio of 1:25; and methanol concentration of 3% (v/v). Expressed AspS.RWR is then concentrated using ultrafiltration method and purified using cation-exchange chromatography in CH-650 resin. SDS-PAGE analysis showed AspS.RWR protein band on ~7,4 kDa; matched with existing literatures. Concentration of AspS.RWR expressed in BMMY is 0,38 mg/mL, while the one expressed in BSM is 0,15 mg/mL. The purification step is continued with AspS.RWR expressed in BMMY. Elution of AspS.RWR using 50 mM citrate buffer pH 5,00 containing 750 mM dan 1000 mM NaCl has resulted in a pure and clean protein band in ~7,4 kDa; after SDS-PAGE analysis. |
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