Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening

Frontiers in Microbiology

Tamara Hillenaar¹  Robert Daniels¹  Henry Ampah-Korsah¹  Jan-Willem de Gier¹  Alexandros Karyolaimos¹  Anna Mestre Borras¹  Katarzyna Magdalena Dolata²  Susanne Sievers²  Katharina Riedel² 

[1] Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden;Institute of Microbiology, University of Greifswald, Greifswald, Germany;

关键词: Escherichia coli;    recombinant protein;    periplasm;    signal peptide;    protein production rate;    protein production screen;   

DOI  :  10.3389/fmicb.2019.01511

来源: DOAJ

【 摘 要 】

Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In E. coli, disulfide bond containing recombinant proteins are often targeted to the periplasm by an N-terminal signal peptide that is removed once it passes through the Sec-translocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.

【 授权许可】

Unknown