(24c) The Culture Environment in Bioreactors Is the Key to the Production and Architecture of Recombinant Protein-Based Nanoparticles in Thermoinduced E. coli

When producing recombinant proteins (RP) in thermoinduced E. coli tend to aggregate intracellularly with endogenous proteins into pseudospherical and mechanically stable nanoparticles known as inclusion bodies (IBs) [1,2]. Recuperation of IBs by cell rupture and centrifugation is considered a pre-purification stage due to their easy recovery process, long-term storage with proteinase resistance, and simplicity in the solubilization and refolding of the RP [1,2,3]. In this work, culture conditions (pH, temperature, and dissolved oxygen) were evaluated in a stirred tank bioreactor to determine the bacterial growth, productivity, and structural characteristics of IBs.

As model E. coli strain W3110 transformed with plasmid pV3-uri200N encoding human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) under the λpL-cI857 system was used [4,5]. All cultures were carried out in triplicate in a 1.0 L stirred tank bioreactor. The online data were acquired, and the pH and DOT sensors were connected to the ADI1010 Biocontroller (Gettinge-Applikon Biotechnology). The growth of E. coli was followed by optical density at 600 nm. The supernatant was used to measure glucose by the Biochemistry Analyzer YSI 2900 (YSI Life Sciences, USA). Organic acids were quantified by HPLC (Shimadzu, Japan). IBs were isolated, as described by Calcines-Cruz et al. [6]. The Bio-Rad Protein Assay was used to determine protein concentration. The RP was estimated by densitometry analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 15% gel-stained. Evaluation of protease resistance of purified IBs was done using proteinase K (PK, Bioline, UK). Unfolding of the IBs was evaluated by solubilization of IBs with GnHCl. Morphology and size were analyzed under TEM following Castellanos-Mendoza et al. [7]. The hydrodynamic diameter of the IBs was determined by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Inst. Ltd, UK).

Recombinant E. coli cultures grown at 34°C showed a ~69% increase in specific growth rate compared to cultures grown at 30°C. Although the induction of the RP begins when it rises to > 38°C, the amount of RP in IBs was significantly higher in cultures grown at 34 °C pre-induction. Moreover, IBs from cultures grown at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. On the other hand, changes as small as 3 °C in post-induction (39 °C vs 42 °C) favor biomass synthesis at 39 °C, but limit RP production compared to 42 °C. In turn, IBs formed at 42°C were less prone to degradation and presented fewer amyloid-like structures related to rapid IBs formation. In comparison, IBs formed slowly at 39 °C presented a higher proportion of amyloid-like structures, more susceptible to degradation. In both thermoinduction scenarios, the IBs were more resistant as the induction time increased.

On the other hand, cultures that control the dissolved oxygen obtain larger biomass than uncontrolled and limited DO. Moreover, DO between 5% and 10% (according to air saturation) post-induction favored the production of rHuGM-CSF and limited the attack of proteases. The lower amyloid content of IBs could also favor solubilization processes, as well as those with elevated levels of structures like native proteins. IBs harvested from cultures controlled after induction at 70% DOT and those without DOT control were less resistant to proteolytic degradation and less stable against chemical solubilization. In addition, they presented smaller sizes than those produced at 5% and 10% of DOT post-induction. This data suggests that those IBs (from 70% DOT and uncontrolled) may be less compact, with a lower proportion of amyloid-like structures than those other IBs typically obtained in industry and academia (among 5 to 30% of DOT). This work presents evidence of the need to know the effect of culture conditions on the production of RP and on the architecture of the IBs. Pre and post-induction temperature and DOT control strategies might play an important role in the IBs' production and in the recovery, refolding, and purification of RP.

References

[1] Valdez-Cruz NA, Trujillo-Roldán MA (2023). In Inclusion Bodies: Methods and Protocols (pp. 17-30). New York, NY: Springer US.

[2] Restrepo-Pineda S, Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA (2021) FEMS Microbiology Reviews, 45(6), fuab023.

[3] de Marco A, Ferrer-Miralles N, Garcia-Fruitós E, Mitraki A, Peternel S,Rinas U, Trujillo-Roldán MA, Valdez-Cruz NA, Vázquez E, Villaverde A (2019) FEMS Microbiol Rev. 43(1): 53-72

[4] Restrepo-Pineda S, Rosiles-Becerril D, Vargas-Castillo AB, Ávila-Barrientos LP, Luviano A, Sánchez-Puig N, García Hernández E, Pérez NO, Trujillo-Roldán MA, Valdez-Cruz NA. Elec J Biotechnol. (2022) 59: 96-106

[5] Restrepo-Pineda S, Sánchez-Puig N, Pérez NO, García-Hernández E, Valdez-Cruz NA, Trujillo-Roldán MA. Appl Microbiol Biotechnol. (2022) 106(8):2883-2902.

[6] Calcines-Cruz C, Olvera A, Castro-Acosta RM, Zavala G, Alagón A, Trujillo-Roldán MA, Valdez-Cruz NA (2018) Int J Biol Macromol. 108:826-36

[7] Castellanos-Mendoza A, Castro-Acosta RM, Olvera A, Zavala G, Mendoza-Vera M, García-Hernández E, Alagón A, Trujillo-Roldán MA, Valdez-Cruz NA. Microb Cell Fact. 2014;13:137

Acknowledgments

Programa de Apoyo a Proyectos de Investigación e InnovaciónTecnológica, Universidad Nacional Autónoma de México” (PAPIIT-UNAM, IN210822 to NAVC, IN211422 and IV201220 to MATR). Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT, CF-2023-I-1248)