A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli

• Published in: Journal of biotechnology 2022-01, Vol.344, p.11-23
• Main Authors: Kupriyanova, Elena V., Sinetova, Maria A., Leusenko, Anna V., Voronkov, Alexander S., Los, Dmitry A.
• Format: Article
• Language: English
• Subjects: Carbonic anhydrase; Carbonic Anhydrases - genetics; Carbonic Anhydrases - metabolism; Crocosphaera subtropica ATCC 51142; Cyanobacteria - enzymology; EcaA; Escherichia coli - genetics; Escherichia coli - metabolism; Heterologous expression; Periplasm - metabolism; Protein secretion; Protein Sorting Signals; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Signal peptide
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2021.12.006

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli. •EcaA is the extracellular carbonic anhydrase of C. subtropica ATCC 51142.•Leader peptide of EcaA ensures efficient secretion of recombinant proteins in E. coli.•Leader peptide of EcaA efficiently functions even as a part of a fusion protein.•The efficiency of EcaA leader peptide is comparable to that of PelB and TorA leaders.