Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris. (25th June 2019)

• Record Type: Journal Article
• Authors: Palanikumar, Indumathi; Katla, Srikanth; Tahara, Nariyasu; Yui, Midori; Zhang, Rui; Ebihara, Akio; Sivaprakasam, Senthilkumar
• Is Part Of: Biotechnology progress. Volume 35:Number 5(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2019-06-25
• Subjects: alcohol oxidase -- high cell density cultivation -- ovine angiotensinogen -- Pichia pastoris; Biotechnology -- Periodicals; Food industry and trade -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/btpr.2866
• Languages: English
• ISSNs: 8756-7938

Abstract

Abstract: Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin‐I, the first product of the angiotensin‐processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high‐level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two‐state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature ( T m ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin‐I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin‐I/min), possibly because of its mannosylated N‐glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.