title: A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in E. coli.
abstract: Human neurturin (NTN) is a cystine knot growth factor with potential therapeutic use in diseases such as Parkinson's and diabetes. Scalable high titer production of native NTN is particularly challenging because of the cystine knot structure which consists of an embedded ring comprised of at least three disulfide bonds. We sought to pursue enhanced scalable production of NTN in Escherichia coli. Our initial efforts focused on codon optimization of the first two codons following AUG, but these studies resulted in only a marginal increase in NTN expression. Therefore, we pursued an alternative strategy of using a bicistronic vector for NTN expression designed to reduce mRNA secondary structure to achieve increased ribosome binding and re-initiation. The first cistron was designed to prevent sequestration of the translation initiation region in a secondary conformation. The second cistron, which contained the NTN coding sequence itself, was engineered to disrupt double bonded base pairs and destabilize the secondary structure for ribosome re-initiation. The ensemble approach of reducing NTN's mRNA secondary structure and using the bicistronic vector had an additive effect resulting in significantly increased NTN expression. Our strain selection studies were conducted in a miniaturized bioreactor. An optimized strain was selected and scaled up to a 100 L fermentor, which yielded an inclusion body titer of 2 g/L. The inclusion bodies were refolded to yield active NTN. We believe that our strategy is applicable to other candidate proteins that are difficult-to-express due to stable mRNA secondary structures. Biotechnol. Bioeng. 2017;114: 1753-1761. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, LLC, 1 MedImmune Way, Gaithersburg, Maryland, 20878.
authors.forename: Varnika
authors.identifier: 0000-0003-1599-3175
authors.initials: V
authors.lastname: Roy
authors.affiliation: Innovative Medicines, AstraZeneca R&D, Mölndal, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Roth
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, LLC, 1 MedImmune Way, Gaithersburg, Maryland, 20878.
authors.forename: Mark
authors.initials: M
authors.lastname: Berge
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, LLC, 1 MedImmune Way, Gaithersburg, Maryland, 20878.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Chitta
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, LLC, 1 MedImmune Way, Gaithersburg, Maryland, 20878.
authors.forename: Sucheta
authors.initials: S
authors.lastname: Vajrala
authors.affiliation: Analytical Biotechnology, MedImmune, LLC, Gaithersburg, Maryland.
authors.forename: Srilatha
authors.initials: S
authors.lastname: Kuntumalla
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, LLC, 1 MedImmune Way, Gaithersburg, Maryland, 20878.
authors.forename: Albert
authors.initials: A
authors.lastname: E Schmelzer
authors.affiliation: Cell Culture and Fermentation Sciences, MedImmune, LLC, 1 MedImmune Way, Gaithersburg, Maryland, 20878.
authors.forename: Ron
authors.initials: R
authors.lastname: Schoner
mesh_terms: D004926:Escherichia coli / Q000502:physiology*; D005091:Exons / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005796:Genes / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D051101:Neurturin / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D013329:Structure-Activity Relationship; D015854:Up-Regulation / Q000235:genetics
chemical_list: D051101:Neurturin; D012333:RNA, Messenger
keywords: E. coli; Micro-24; bicistronic vector scalable production; cystine knot; destabilization; higher titer; mRNA secondary structure; neurturin