title: Efficient Production of Hydroxylated Human-Like Collagen Via the Co-Expression of Three Key Genes in Escherichia coli Origami (DE3).
abstract: Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and D-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.
authors.affiliation: Key Laboratory of Biomass Chemical Engineering (Ministry of Education), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
authors.forename: Yunping
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Shangdong Jincheng Biopharmaceutical Corporation Limited, Zibo, 255130, People's Republic of China.
authors.forename: Xiuliang
authors.initials: X
authors.lastname: Yang
authors.affiliation: Key Laboratory of Biomass Chemical Engineering (Ministry of Education), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
authors.forename: Baojian
authors.initials: B
authors.lastname: Hang
authors.affiliation: Shangdong Jincheng Biopharmaceutical Corporation Limited, Zibo, 255130, People's Republic of China.
authors.forename: Jiangtao
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Biomass Chemical Engineering (Ministry of Education), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: Key Laboratory of Biomass Chemical Engineering (Ministry of Education), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
authors.forename: Feng
authors.initials: F
authors.lastname: Huang
authors.affiliation: Key Laboratory of Biomass Chemical Engineering (Ministry of Education), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China. znxu@zju.edu.cn.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D019149:Bioreactors; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D060847:Metabolic Engineering; D011392:Proline / Q000378:metabolism; D064828:Prolyl Hydroxylases / Q000096:biosynthesis / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D003094:Collagen; D011392:Proline; D013401:Sugar Alcohol Dehydrogenases; C090492:D-arabinonolactone oxidase; D064828:Prolyl Hydroxylases
keywords: E. coli; Human like collagen; Hydroxylation; Post-translation modification; Prolyl 4-hydroxylase