A bicistronic vector with destabilized mRNA secondary structure yields scalable higher titer expression of human neurturin in E. coli. Issue 8 (18th May 2017)

• Record Type: Journal Article
• Authors: Roy, Varnika; Roth, Robert; Berge, Mark; Chitta, Rajesh; Vajrala, Sucheta; Kuntumalla, Srilatha; E. Schmelzer, Albert; Schoner, Ron
• Is Part Of: Biotechnology and bioengineering. Volume 114:Issue 8(2017)
• Page Start: 1753
• Page End: 1761
• Publication Date: 2017-05-18
• Subjects: neurturin -- cystine knot -- mRNA secondary structure -- destabilization -- bicistronic vector scalable production -- higher titer -- E. coli -- Micro‐24; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26299
• Languages: English
• ISSNs: 0006-3592

Abstract

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. Abstract : This study describes a bicistronic vector approach to reduce mRNA secondary structure and enhance expression of Neurturin (NTN) in E. coli . The first cistron prevents sequestration of the translation initiation region in a secondary conformation leading to increased ribosome binding. The second cistron coding NTN, was engineered to destabilize secondary structure for ribosome re‐initiation. The ensemble approach of reducing NTN's mRNA secondary structure and using a bicistronic vector had an additive effect resulting in significantly increased NTN expression.