THE ROLE OF SPACER AND LINKER PEPTIDES IN PROINSULIN ASPART SECRETION IN Pichia Pastoris AND CHARACTERIZATION OF PROINSULIN ASPART

• Main Author: Asri Kurniatin, Popi
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/42671
• Institution: Institut Teknologi Bandung

Insulin is a peptide hormone used in diabetes therapy. It comprises of two polypeptide chains, connected by two interchain disulfide bonds and one intrachain disulfide bond. Insulin has been modified to insulin analog to meet the therapeutic requirement. One of the insulin analog that is widely used in Indonesia is insulin Aspart. Insulin Aspart is a fast-acting insulin analog where amino acid of B28 has been substituted from proline to aspartic acid, resulting in the increase of repulsive force between chains. Hence, the dimer formation of insulin is inhibited, leading to a rapid absorption of insulin. Insulin is mainly produced as a proinsulin in which the B chain is connected to the A chain by a peptide linker. Successful production of extracellular proinsulin in P. pastoris is determined by many factors, i.e. specific signal peptide for efficient secretion, the design involving the use of proinsulin linker, the strain of P. pastoris, and production conditions. The ?-MF signal peptide originating from Saccharomyces cerevisiae is widely used for proinsulin secretion in Pichia pastoris. The signal peptide consists of a spacer EAEA connecting it to the proinsulin. However, the disadvantage of proinsulin secretion using ?-MF signal peptide is the incomplete cleavage of the EAEA spacer resulting in mixture forms of proinsulins with the addition of amino acids at the N-terminal protein. Meanwhile, deletion of this spacer decreases expression level to 54%. The addition of spacer peptide EEAEAEAEPK, instead of EAEA, successfully enhances the production of proinsulins in P. pastoris. Unfortunately, the extended spacer peptide is partially degraded by yeast aspartyl protease 3, producing mixture forms of proinsulins with various molecular weight. Furthermore, the selection of linker in the design of proinsulins influences the expression level of proinsulins. The most widely used linkers in proinsulins are AAK and RWR. The factors that can be optimized to obtain high level production of proinsulin are methanol induction, cell density, and oxygen supply. In addition, proinsulin production in various P. pastoris strains show different yields. The purposes of this research are to study the effect of spacer and linker peptides in the secretion of proinsulin Aspart in P. pastoris, the optimum conditions of proinsulin Aspart production, the types of P. pastoris strains for proinsulin Aspart production, and to characterize the proinsulin Aspart. The research was divided into three main studies, the first one is to study the secretion of proinsulin Aspart with linker variety of RWR and AAK using ?-MF signal peptide without spacer and ?-MF signal peptide with the EEAEAEAEPK spacer. The second stage is to examine the factors that influence the production of proinsulin Aspart including methanol concentration for induction, aeration, cell density, and variety of strains. Finally, the characterization of the proinsulin Aspart; molecular weight, glycosylated forms of proinsulin Aspart, and disulfide bond. Characterization was carried out by the methods of RP-HPLC, LCMS/MS, ESI-MS. Proinsulin Aspart with the linker RWR is successfully secreted using ?-MF signal peptide without spacer. Meanwhile, ?-MF signal peptide with spacer EEAEAEAEPK did not increase the expression level of proinsulin Aspart. The Western Blot analysis and ESI-MS show an extracelullar proinsulin Aspart with a size of 6.3 kDa. Whereas the respective molecule was not found in the intracellular fluid. This finding suggests that the cleavage of signal peptide is not determined by spacer peptides. Therefore, the spacer peptide is not essential for proinsulin Aspart secretion in P. pastoris. Proinsulin Aspart constitutes of linker RWR is optimally expressed in P. pastoris KM71 using ?-MF signal peptide without spacer. The proinsulin Aspart is optimally produced by 2% (v/v) of methanol induction for two days at initial OD600 of ~60, and aeration (ratio of culture volume to flask volume) of 1:25. The molecular weight of proinsulin Aspart is 6.3 kDa, presumably containing three disulfide bonds, and the glycosylated form of proinsulin Aspart is not observed. The use of ?-MF signal peptide without a spacer for the secretion of proinsulins is very beneficial because it only produces one type of proinsulin Aspart with a molecular weight of 6.3 kDa. The use of RWR linker in the design of Aspart proinsulins is able to increase expression levels. The production of proinsulins in P. pastoris generally uses GS115 and X33 strains. In this study, the expression of the proinsulin Aspart shows the best results in P. pastoris KM71 strain. The advantage of the KM71 strain is that it requires lower amounts of methanol induction than GS115 and X33 strains.