Journal
CURRENT PHARMACEUTICAL BIOTECHNOLOGY
Volume 15, Issue 9, Pages 856-863Publisher
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/1389201015666141012182106
Keywords
Bioactivity; brain natriuretic peptide; human serum albumin; fusion number; fusion orientation; Pichia pastoris
Funding
- Natural Science Foundation of Jiangsu Province [BK2012104]
- Public Service Platform for Science and Technology Infrastructure Construction Project of Jiangsu Province [BM2012066]
- China National Natural Science Foundation [81273437]
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Human brain natriuretic peptide (BNP) is utilized in the treatment of acute decompensated congestive heart failure. However, BNP has limited clinical use owing to its rapid clearance and the need for continuous intravenous infusion. Thus, we generated human serum albumin (HSA)-BNP fusion constructs to produce long-acting fusion proteins in Pichia pastoris. Four fusion proteins, BNP-HSA, (BNP)(2)-HSA, (BNP)(4)-HSA, and HSA-(BNP)(2), were constructed, with different numbers of BNP molecules and fusion orientations. BNP-HSA was most abundantly expressed in Pichia pastoris and (BNP)(4)-HSA had the lowest yield, probably because of the high copy number of BNP. Western blot analysis confirmed the immunogenicity of both BNP and HSA for the four fusion proteins. A cGMP activity assay was used to measure the ability of fusion proteins to stimulate intracellular cGMP expression. Results showed that the fusion protein HSA-(BNP)(2) activated human natriuretic peptide receptor A (hNPR-A) with potency similar to that of BNP, despite using a 10-fold higher dosage than BNP. The other three fusion proteins (BNP-HSA, (BNP)(2)-HSA, and (BNP)(4)- HSA), only slightly increased NPR-A activity. In addition, fusion with HSA successfully prolonged BNP bioactivity, stimulating intracellular cGMP expression over 24 h. In conclusion, HSA-(BNP)(2), with two BNP molecules fused at the C-terminus of HSA, has the highest and most prolonged BNP bioactivity in activating human NPR-A.
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