We developed large-scale preparation of phycoerythrin from Porphyra haitanensis, a main economic red algae in China. Firstly, P. haitanensis thallus was broken by using "swelling and smash" method. Then times of grads ammonium sulfate precipitation applied to the crude extraction were compared. Desalted solution was further purified with one-step chromatography using hydroxyapatite and properties on spectrum and molecular weight were identified finally. The results indicated that after four times of ammonium sulfate precipitation (15%, 50%, 10% and 40%), the absorption spectrum purity of P. haitanensis achieved 0.9 (A564/A280), and 507.82 mg phycoerythrin (A564/A280 > 3.2) was obtained from 7 kg fresh algae after further hydroxyapatite chromatography. This research provides a potential way for preparation of phycoerythrin in large sclae.
Ammonium Sulfate ; chemistry ; Chromatography ; methods ; Phycoerythrin ; isolation & purification ; Porphyra ; chemistry

Animals ; Biocatalysis ; Cattle ; Enteropeptidase ; chemistry ; metabolism ; Humans ; Models, Molecular ; Oryzias ; metabolism ; Structure-Activity Relationship ; Substrate Specificity

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.
Atrial Natriuretic Factor ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Gene Expression ; Humans ; Metalloendopeptidases ; Peptides ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis