Human coagulation factor IX is a 57kDa plasma serine protease made in Liver which plays a vital role in the blood coagulation cascade. FIX deficiency causes severe disorder Hemophilia B or Christmas disease. Nowadays, recombinant proteins have important roles in treatment of diseases. Although, cultivated mammalian cells because of their ability for producing properly folded protein molecules, have become a dominant system for the production of recombinant proteins with medical application. There are several kinds of methods to optimization of gene expression but one of the most useful method is codon optimization. Codon optimization is adaptation of nucleic acid sequences according to host cells codon usage. The replacement of rare codons with the frequent codons of host cells could enhance the expression of interested protein. Altering the coding sequence to increase protein expression is highly cost-effective. The aim of this study is expression and production of clotting FIX in CHO cells and eventually increasing the expression level based on codon usage host cells.
Materials and methods
The DNA sequences of the open reading frame (ORF) for the FIX protein have been well adapted to the codon usage of CHO cells. First of all this sequence has been cloned into pCDNA3.1+ expression vector, then the construct was transfected into the CHO cells with electroporation method. Later, the protein production was confirmed by SDS-PAGE, Western blotting.
The exact bond of FIX has not been detected in SDS-PAGE due to the abundance of albumin, in contrast the Western blotting results of the cells supernatant confirmed the expression and secretion of FIX protein from cells.
According to the above-mentioned, it has showed acceptable level of protein expression in SDS-PAGE and western blotting. So we will analysis data by ELISA technique.