Secretory Expression And Scale-up Production Of The Recombinant Human Thyroid Peroxidase

Human thyroid peroxidase(hTPO)is an essential enzyme in thyroid cells to synthesize thyroid hormones and can be used to detect antibodies in the sera of patients with a number of autoimmune thyroid disorders.Therefore,the expression and production of recombinant hTPO with immune reactivity is of great significance for the development of autoimmune thyroid desease diagnostic products.It is well known that the baculovirus expression vector system(BEVS)has a very prominent advantage in expressing the human active protein,and it has been widely used in the expression of antigen proteins associated with various diseases.hTPOt has also been expressed in the BEVS,but the main problem is that only a small amount of protein is transported to the cell culture supernatants through the secretion pathway,and most of recombinant proteins accumulates in the cell lysis precipitates,which greatly increases the complexity of protein purification.In this work,we established a process to improve the expression of truncated form hTPO(hTPOt)in BEVS.(1)Firstly,5 secretion signal peptides were selected to replace the original N-terminal secretion signal peptide.Among them,the secretion signal peptide frompeptidyl-glycine alpha-amidating monooxygenase,known as SPPAM was found to be effective in assisting the secretion of hTPOt in High 5 cells.(2)Secondly,to achieve the scale-up production of p-hTPOt protein,we tried to express p-hTPOt in a Wave-like bioreactor,and optimized the parameters:rotational speed,rotation angle,cell inoculation density and serum addition.Based on the optimized expression conditions,we amplify the production of p-hTPOt in a 5 L scale.The harvested supernatants were purified by immobilized metal affinity chromatography(IMAC)and ion exchange chromatography,and the purity of the harvested proteins reached 95%.(3)Finally,the purified p-hTPOt were tested with human serum samples by ELISA,and showing the high sensitivity and specificity(80%and 100%).Collectivly,we established an optimized procedure for producing purified hTPOt based on BEVS and achieved scale-up production in the Wave-like bioreactor,which could provide cheap and efficient antigen materials for hTPO-related research or development.