Enhancing extracellular production of recombinant proteins in Escherichia coli by co-expressing with a haloarchaeal protein containing a putative LolA-like domain

• Published in: Applied microbiology and biotechnology 2021-06, Vol.105 (11), p.4609-4620
• Main Authors: Wang, Jian, Hao, Chuang, Cao, Lei, Yao, Yitong, Ding, Yidi, Yang, Yong, Tang, Xiao-Feng, Tang, Bing
• Format: Article
• Language: English
• Subjects: Archaeabacteria; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Cell culture; Deletion; Domains; E coli; Escherichia coli; Galactosidase; Life Sciences; Lysis; Lysozyme; Membrane permeability; Membranes; Methods; Microbial Genetics and Genomics; Microbiology; N-Terminus; Natrinema; Peptides; Periplasm; Permeability; Physiological aspects; Production processes; Proteins; Recombinant proteins; Subtilase; β-Galactosidase
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-021-11352-5

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by β-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli . A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli . Key points • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.