Judul: Kloning dan Ekspresi Human Interferon-alfa2a pada Yeast Metilotropik Pichia pastoris
Abstrak: Interferon (IFN) is a group of proteins that are secreted by body cells as a result of exposure to biological vertebrates such as: viruses, bacteria, protozoa and other compounds. There are three types of interferon, namely: alpha (α), beta (β) and gamma (γ) IFN. Interferon-α2a belongs to the type I interferon. This protein has a molecular weight of 21.550 kDa and consists of 188 amino acids (23 amino acid of signal peptide and 166 amino acids of mature protein). Interferon-α2a is also known to be used for the treatment of hepatitis C and as anti-tumor and antivirus agents. Regulator gene for IFN production under normal conditions, is in the off position to IFN is not produced. But when there are external stimuli, both viral and bacterial infections, a regulator gene switches on, and IFN production system running. At the time of an attack from a variety of disease agents actually the body will produce IFN, but generally the amount produced is not sufficient to fight the disease agents that multiply very quickly. Therefore IFN intake from outside is needed. This is the beginning of the use of IFN as a drug. Genetic engineering is an activity to manipulate the genes to get new products by creating recombinant DNA through gene insertion. Recombinant DNA technology has been used to produce IFN. Yeast such as Pichia pastoris can be an alternative as the host (system) to express human interferon genes and as a producer of IFN protein. Expression system P. pastoris offers several advantages for the production of recombinant proteins, such as: the high level expression of recombinant proteins, ease of scaling-up fermenter, low cost and ease of transformation techniques and selection of transformed cells such as bacteria . The existence of inducible AOX1 promoter is very strong, easily control the expression of recombinant proteins by induction. AOX1 is a promoter that controls gene expression of alcohol oxidase to metabolize methanol as carbon source of P. pastoris. Therefore,this study aimed to clone human interefron-α2a (hifnα2a) gene in E. coli and express it in metylotrophic yeast P. pastoris. Vector used in this study is pPICZαB (Invitrogen). Ligation process between insert (hifn-α2a gene) and vector (pPICZαB) performed by using T4 DNA ligase, which functions synthesize the formation of phosphodiester bonds linking the nucleotides with nucleotide insert plasmid vectors to produce recombinant (pPICZαB-hifnα2a). This recombinant plasmid transformed into XL1 blue cells (E. coli) by Heat shock method. The results of sequencing analysis (clone 6) using 5'AOX and 3'AOX primers indicate that DNA base sequence of human IFN-α2a genes has been subclone into pPICZαB vector (in frame). Recombinant plasmid (clone 6) linearized using BstX1 enzyme before transformed into X33 yeast cell. Transformation into cell was done by Electroporation. The X33 cell P. pastoris strain was used to express human interferon-α2a. X33 is strains with the phenotype Mut+ (methanol utilization plus) and can be used for positive transformants selection using zeocin. Phase protein production work using Mut+ strain occurred in two phases. The first phase is the use of glycerol to obtain a certain amount of biomass (high cell
Keyword: