Increasing Recombinant Protein Production in E. coli via FACS-Based Selection of N-terminal Coding DNA Libraries

Data for a manuscript to be deposited to the FEBS Journal. Abstract as deposited: We present a novel approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated a profound impact of a few nucleotides following the initiator codon (further to as N-terminal sequences) on their expression. Most of these investigations have been limited to selecting from a few rationally designed sequences. In contrast we used directed evolution-based methodology screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increasing expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.投稿至《FEBS Journal》的手稿配套数据集。 已提交的手稿摘要如下： 本研究提出一种改造重组蛋白N端序列的全新方法，以提升其在大肠杆菌（Escherichia coli）中的表达产量。既往研究已证实，起始密码子下游的少数核苷酸（以下简称N端序列）对蛋白表达具有显著影响，但此类研究大多仅局限于从少量理性设计的序列中进行筛选。与之不同，本研究采用基于定向进化的方法，从编码目标蛋白N端的DNA文库中筛选海量多样化序列。为便于筛选表达量提升的靶标工程菌，我们将绿色荧光蛋白（GFP）基因克隆至靶基因的C端，并通过荧光激活细胞分选术（FACS）基于荧光信号分离细胞。通过该系统化实验流程，我们成功将多种构建体的可溶性重组蛋白产量提升至原水平的30倍以上。