Secreted Expression Of Recombinant Human Brain Natriuretic Peptide In Pichia Pastoris

Brain Natriuretic Peptide (BNP) is a genetically distinct member of the natriuretic peptide family, which also consists of atrial natriuretic peptide (ANP) of cardiac myocyte origin and C-type natriuretic peptide (CNP) of central nervous , endothelial, and renal epithelial cell origin. Althoμgh originally extracted from porcine brain, BNP is now thoμght to be principally of cardiac myocyte origin.BNP is a cardiac hormone that regulates blood pressure and fluid homeostasis. Under physiological conditions, BNP is produced by atrial and ventricular cardiomyocytes. BNP is released mainly in response to increased stretch or wall-tension and is broadly involved in the regulation of blood pressure, blood volume and sodium balance. The actions are performed by natriuresis, vasodilatation, and inhibition of the renin–angiotensin–aldosterone axis and the sympathetic nervous system. The plasma concentration of BNP is raised in patients with cardiac disease, particularly those with heart failure.Recent studies in experimental and human congestive heart failure (CHF) have focused on BNP, on the basis of its greater natriuretic action and itssuperiority as a clinical biomarker for altered myocardial function and structure. BNP is highly sensitive and fairly specific markers for diagnosing CHF and to detect left ventricular dysfunction. BNP Level is related to the severity of disease and strongly associated to outcome in patients with heart failure. It has also been found to be related to the subsequent response to treatment and has been sμggested to be useful tools to guide treatment in heart failure patients. So BNP is currently used clinically to aid the diagnosis of CHF, assessing the severity of CHF and risk stratification in patients with coronary artery diseases.The methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisine, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit.Therefore several aspects of research work were reported in this paper:1. Design and synthesis of hBNP geneWe change the genetic codons of mature peptide to Pichia pastoris favorite codons by artificial synthesis and insert XhoI target site,Kex2 in 5'end, insert terminal codons taa and EcoRI target site in 3' end. Kex2 is essential to cutαsignal peptide efficiently. Cloning vector is pUC57, cloning site is XhoI/EcoRI, host bacterium is DH5α.Inoculate some host bacterium in LB medium containing Amp, shake in 37℃overnight, then extract synthetic recombinant plasmid pUC57-hBNP by Plasmid extraction Kit in accordance with directions.2. Construction of recombinant plasmid pPICZαC-hBNPRecombinant plasmid pUC57-hBNP is cut by XhoI/EcoRI, after purification it was inserted into the pPICZαC vector which is cut by the same enzymes. After confirmation by endonuclease digestation assay and sequencing, recombinant vector was linearizated by SacI, and then transformed into Pichia pastoris X-33 via electrotransformate.3. Screening of recombinant Pichia pastoris strainAfter electrotransformation yeast fungus was spread on YPD liquid medium containing Zeocin, culturing in 28℃for about 72h. Then we extracted the genomic DNA of the transformed yeasts via boiling-frozen-boiling method to perform PCR using the AOX1 5' and AOX1 3' primers.4. Expression and assay of rhBNPProliferated the PCR tested positively yeast clones, then induced the expression of rhBNP with 0.5% methanol in BMMY media. Analyzed the expression products by SDS-PAGE. The results indicated that there was a 3500Da protein in the supernatant.In conclusion, our studies succeed in secreted expression of hBNP in Pichia pastoris. we discovered there was a 3500Da protein in the supernatantVia SDS-PAGE, and screen the positively transformed Pichia pastoris strain with steady expression capability.