Abstract
Human C-reactive protein (CRP), a classical human acute-phase plasma protein, is not only a sensitive systemic inflammatory marker but also an independent risk predictor of cardiovascular diseases. However, existing heterologous expression systems for expressing CRP is not efficient and cost-effective for large-scale industrial production of CRP to meet the growing market demand for CRP. This study aims to improve the secretion of recombinant CRP by Pichia pastoris via optimizing signal peptides, promoters and carbon sources. The CRP genes with encoding four different signal peptides were designed and synthesized. The genes were cloned into pPICZαA or pPICZ B, respectively via splicing by overlap extension polymerase chain reaction (SOE-PCR) technology and expressed in P. pastoris X-33, regulated by the alcohol oxidase I promoter (pAOX1). The CRP led by the α-mating factor secretion signal peptide (α-MF) was secreted at the highest level in these signal peptides. Then, a constitutive construct and expression of the CRP genes were achieved by switching to the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). Subsequently, different carbon sources and at different concentrations were used to further improve the secretion of CRP. The expression of CRP with the α-MF driven by the pGAP gave the highest yield of secreted CRP, about 3mg/l of culture on the optimized culture conditions. The purified recombinant CRP exhibited good immunoreactivity determined by ELISA with anti-human CRP monoclonal antibody. The efficient engineering strategy established in this work might provide potential uses in large-scale industrial production of human CRP in the future.
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