learColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purificatio

Author(s)

Cardoso, Valdemir M. ^[1] ; Paredes, Sheyla A. H. ^[1] ; Campani, Gilson ^{[2, 1]} ; Goncalves, Viviane M. ^[3] ; Zangirolami, Teresa C. ^[1]

Affiliation

^[1] Fed Univ Sao Carlos UFSCar, Dept Chem Engn, Grad Program Chem Engn PPGEQ, Rodovia Washington Luis, Km 235, BR-13565905 Sao Carlos, SP - Brazil

^[2] Univ Fed Lavras, Dept Engn, BR-37200900 Lavras, MG - Brazil

^[3] Butantan Inst, Lab Vaccine Dev, Av Vital Brasil 1500, BR-05503900 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Applied Microbiology and Biotechnology; v. 106, n. 3, p. 1011-1029, FEB 2022.
• Web of Science Citations: 0

Abstract

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi (R) BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 degrees C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-beta-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 degrees C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. (AU)

• FAPESP's process: 16/50413-8 - Pulmonary delivery of a targeted mucosal nanocarrier vaccine for pneumonia
• Grantee: Viviane Maimoni Gonçalves
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/10291-8 - Process intensification and integration for pneumococcal surface protein A production and purification
• Grantee: Teresa Cristina Zangirolami
• Support Opportunities: Regular Research Grants