Increasing recombinant protein production in E. coli via FACS‐based selection of N‐terminal coding DNA libraries

• DOI: 10.1111/febs.17376
• Publication Date: 2024-12-27T05:33:26Z
• Authors: Štěpán Herynek; Jakub Svoboda; Maroš Huličiak; Yoav Peleg; Ľubica Škultétyová; Pavel Mikulecký; Bohdan Schneider
• Keywords: Cell Sorting; Coding region

Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following start codon can significantly influence protein expression. However, impact these is construct-specific and not universally applicable all proteins. Most previous been limited selecting from few rationally designed sequences. In contrast, used directed evolution-based methodology, screening large numbers diversified derived DNA libraries coding for N-termini investigated To facilitate identification cells with increased expression target construct, cloned GFP gene at C-terminus expressed genes fluorescent activated cell sorting (FACS) separate based on fluorescence. By this systematic workflow, successfully elevated soluble multiple constructs up over 30-fold.