title: Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris.
abstract: 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.
authors: Department of Clinical Laboratory, The Linzi Peoples' Hospital of Zibo, Shandong, China.
authors: Y M
authors: YM
authors: Li
authors: Department of Clinical Laboratory, Yantai Economic and Technological Development Zone Hospital, Yantai, Shandong, China.
authors: D J
authors: DJ
authors: Li
authors: Department of Ophthalmology, Qingdao Fifth Peoples' Hospital, Qingdao, Shandong, China.
authors: X J
authors: XJ
authors: Xu
authors: Research Center, AI DE Diagnostic Co., Ltd., Qingdao, Shandong, China.
authors: M
authors: M
authors: Cui
authors: Research Center, AI DE Diagnostic Co., Ltd., Qingdao, Shandong, China.
authors: H H
authors: HH
authors: Zhen
authors: Department of Clinical Laboratory, The Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong, China Wangqing0533@aliyun.com.
authors: Q
authors: Q
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D055316:Cystatin C / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: C525030:CST3 protein, human; D003062:Codon; D055316:Cystatin C; D011994:Recombinant Proteins