Production of Recombinant Protein, Human Stem Cell Factor, Using Insect Cell Line

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.