title: Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies.
abstract: Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up.
authors: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, PR China.
authors: Mingzhi
authors: M
authors: Zhao
authors: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, PR China; Life Science College, Southwest Forestry University, Kunming 650224, PR China.
authors: Feilin
authors: F
authors: Wu
authors: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, PR China; Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, PR China; Anhui Medical University, Hefei 230032, Anhui, PR China. Electronic address: xuping_bprc@126.com.
authors: Ping
authors: P
authors: Xu
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019149:Bioreactors; D003001:Cloning, Molecular / Q000379:methods; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D002479:Inclusion Bodies / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014362:Trypsinogen / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014362:Trypsinogen; D014357:Trypsin
keywords: Acetylation; Activation; Cationic trypsin; Purification; Recombinant expression; Refolding optimization