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Xingtai, China

Xu X.J.,Qingdao Fifth Peoples Hospital | Cui M.,AI de Diagnostic Co. | Zhen H.H.,AI de Diagnostic Co. | Wang Q.,Qingdao University
Genetics and Molecular Research

Human cystatin C (CysC) is a cysteine proteinase inhibitor with many potential applications. To facilitate further studies of the functions and applications of CysC, we improved the heterologous expression of CysC using a basic codon optimization method. In this study, we cloned the high-GC content wild-type sequence of the CysC gene and also designed a slightly AT-biased sequence, with codons optimized for expression in the Pichia pastoris GS115 strain. Our results showed that the optimized coding sequence of human CysC increased the expression and secretion of the CysC protein by approximately 3- to 5-fold (90-96 mg CysC/L) in yeast, compared with the expression levels of the native CysC gene (17.9-18.4 mg CysC/L). We designed, constructed, and applied an optimized version of the CysC gene for the Pichia expression system. Our results demonstrate that the optimized coding sequence provides a higher yield of secreted CysC than that produced using the wild-type gene. Our data also serve as a practical example demonstrating a rational design strategy for the heterologous expression of secreted proteins. © FUNPEC-RP. Source

Wang Q.,Qingdao University | Mei C.,AI de Diagnostic Co. | Zhen H.,AI de Diagnostic Co. | Zhu J.,AI de Diagnostic Co.
Journal of Biomedicine and Biotechnology

Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the human cystatin C (cysC) gene using the preferred codons of the prokaryotic system may significantly increase cysC expression in Escherichia coli (E. coli). Specifically, cysC expression may be increased by removing unstable sequences and optimizing GC content. According to E. coli expression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimized cysC (co-cysC) and wild-type cysC (wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid into E. coli BL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni2+-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase in cysC expression in E. coli expression produced by codon optimization techniques may be applicable to commercial production systems. © 2012 Qing Wang et al. Source

Wang Q.,Qingdao University | Wang X.,Institute for Population and Family Planning | Zhang J.,AI de Diagnostic Co. | Song G.,Qingdao University
Experimental and Therapeutic Medicine

In the present study, we standardized a TaqMan locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) probe for the accurate quantification and detection of hepatitis B virus (HBV) DNA in serum (plasma), and evaluated its meth odology. LNA probe technology had a much better detection performance in HBV DNA than the common TaqMan probe. The assay based on the LNA probe had a wider linear detection range, higher sensitivity, stability and amplification efficiency, and a lower concentration of probes than the TaqMan probe. Among the 15 cases with chronic hepatitisB surface antigen (HBsAg) (+) alone, only 4 cases that were detected by TaqMan real-time PCR were negative; however, the same samples were positive by LNA real-time PCR (p<0.05). A positive correlation between viral load measurements for the 35samples with HBV-positive DNA was detected in both LNA and TaqMan real-time PCR. Source

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