Cloning and Expression of Human Interferon alpha 2a in the methylotrophic Yeast Pichia pastoris

Abstract

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 density). The second phase is the reduction of glycerol and methanol for recombinant protein biosynthesis. The level expression of hifn-α2a was identified using Dot blot, SDS PAGE and Western blot. Dot blot is a serological test, which is specific for the detection of reactions between antigens and antibodies, and suitable to identify many types of protein samples. Moreover, SDS PAGE can provide information about the size (MW) of IFN protein by comparing the resulting bands marker size in kiloDalton (kDa) (Figure 12), while Western blot identify specific antibodies to proteins that have been separated. Protein expression levels can be seen by comparing the bands of protein with the control (Figure 13). The existence of protein pattern on the size of 24.050 kDa showed that the protein is secreted into the medium by P. pastoris is hIFN-α2a. These results showed that hifn-α2a gene has been succesfully cloned in E. coli and expressed in P. pastoris.