2.1 Interferon-γ2.1.1 IntroductionInterferons are the family of cytokines whose main role is to inhibit viral infection and intracellular bacterial infection and also tumor control ( Ebrahimi et al., 2012). Interferons are classified into two types based on receptor binding and sequence homology. The primary role of type I interferon (IFN-α, IFN-β, IFN-ω, IFN-τ) is to make cells resistant to viral infections and type II interferon (IFN-γ) is to to regulate the overall immune response (Schroder et al., 2004). IFN-γ is a glycosylated secretory protein with a total molecular weight of 25 kDa. It is one of the main cytokines that regulate both innate and adaptive immunity (Savana et al., 2009). IFN-γ is secreted by CD4+ T helper type 1 (Th1) lymphocytes, cytotoxic CD8+ T lymphocytes, NK cells, and NKT cells, and also by antigen-presenting cells such as macrophages, dendritic cells, and B lymphocytes (McLaren and Ramji, 2009). IFN-γ production is further stimulated by the combination of Interleukin-12 (IL-12) and Interleukin-18 (IL-18), while it is negatively regulated by Interleukin-4 (IL-4), Interleukin-10 (IL- 10), growth promoting factor β, glucocorticoids (Schroder et al., 2004). IL-12 promotes the differentiation of naïve CD4+ T cells into Th1 cells, which produce IFN-γ and enhance the cell-mediated immune response (O'Shea et al., 2002).2.1.2 Gene structureThe IFN-γ gene is located on human chromosome 12 of gene size 501bp (Bureau et al., 1995). Unlike IFN-α and IFN-β, IFN-γ has no introns (Taya et al., 1982). 2.1.3 Protein structure IFN-γ is a homo-dimeric glycoprotein (Savana et al., 2009), of 166 amino acids, in which the 20 N-terminal sequence acts as signal peptides (Taya et al., 1982). It has two subunits and each subunit has 6 alpha helices held together...... middle of paper ......Drocourt et al., 1990).2.2.5 Signal sequenceThe heterologous signal sequence preprot (269bp) of the S. cerevisiae alpha mating factor is often used to induce Sec61p-mediated protein translocation into the endoplasmic reticulum of P. pastoris. Kex2p/Ste13p-mediated processing of the propeptide in this S. cerevisiae sequence is often problematic in P. pastoris, resulting in the production of several amino acids at the N-terminus of the heterologous protein (De Schutter et al., 2009). Processing of the α-factor secretion signal sequence in pPICZα A occurs in two steps, namely preliminary cleavage of the signal sequence by the KEX2 gene product, with the final cleavage of KEX2 occurring between arginine and glutamine in the sequence Glu-Lys-Arg-Glu-Ala-Glu-Ala. The Glu-Ala repeats are further cleaved by the STE13 gene product (Brake et al., 1984).