The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli.

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew 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. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs. KEY POINTS: • Pre-induction temperature determines inclusion bodies architecture • In pre-induction (above 34 °C), the heat shock response increases recombinant protein production • Inclusion bodies at higher pre-induction temperature show a lower amyloid content.

Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights.

Recombinant protein (RP) production from Escherichia coli has been extensively studied to find strategies for increasing product yields. The thermoinducible expression system is commonly employed at the industrial level to produce various RPs, which avoids the addition of chemical inducers, thus minimizing contamination risks. Multiple aspects of the molecular origin and biotechnological uses of its regulatory elements (pL/pR promoters and cI857 thermolabile repressor) derived from bacteriophage λ provide knowledge to improve the bioprocesses using this system. Here, we discuss the main aspects of the potential use of the λpL/pR-cI857 thermoinducible system for RP production in E. coli, focusing on the approaches of investigations that have contributed to the advancement of this expression system. Metabolic and physiological changes that occur in the host cells caused by heat stress and RP overproduction are also described. Therefore, the current scenario and the future applications of systems that use heat to induce RP production are discussed to understand the relationship between the activation of the bacterial heat shock response, RP accumulation and its possible aggregation to form inclusion bodies.

Integrative overview of antibodies against SARS-CoV-2 and their possible applications in COVID-19 prophylaxis and treatment.

SARS-CoV-2 is a novel ß-coronavirus that caused the COVID-19 pandemic disease, which spread rapidly, infecting more than 134 million people, and killing almost 2.9 million thus far. Based on the urgent need for therapeutic and prophylactic strategies, the identification and characterization of antibodies has been accelerated, since they have been fundamental in treating other viral diseases. Here, we summarized in an integrative manner the present understanding of the immune response and physiopathology caused by SARS-CoV-2, including the activation of the humoral immune response in SARS-CoV-2 infection and therefore, the synthesis of antibodies. Furthermore, we also discussed about the antibodies that can be generated in COVID-19 convalescent sera and their associated clinical studies, including a detailed characterization of a variety of human antibodies and identification of antibodies from other sources, which have powerful neutralizing capacities. Accordingly, the development of effective treatments to mitigate COVID-19 is expected. Finally, we reviewed the challenges faced in producing potential therapeutic antibodies and nanobodies by cell factories at an industrial level while ensuring their quality, efficacy, and safety.

Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli: the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies.

The heat-inducible expression system has been widely used to produce recombinant proteins in Escherichia coli. However, the rise in temperature affects cell growth, activates the bacterial Heat-Shock Response (HSR), and promotes the formation of insoluble protein aggregates known as inclusion bodies (IBs). In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis), compared with cultures grown at 30 °C (without induction). Also, the expression of the major E. coli chaperones (DnaK and GroEL) was analyzed. We found that almost twice maximum biomass and rESAT-6 production were obtained in bioreactors (~ 3.29 g/L of biomass and ~ 0.27 g/L of rESAT-6) than in shake flasks (~ 1.41 g/L of biomass and ~ 0.14 g/L of rESAT-6) when induction was carried out at 42 °C, but similar amounts of rESAT-6 were obtained from cultures induced at 39 °C (~ 0.14 g/L). In all thermo-induced conditions, rESAT-6 was trapped in IBs. Furthermore, DnaK was preferably expressed in the soluble fraction, while GroEL was present in IBs. Importantly, IBs formed at 39 °C, in both shake flasks and bioreactors, were more susceptible to degradation by proteinase-K, indicating a lower amyloid content compared to IBs formed at 42 °C. Our work presents evidence that the culture scale and the induction temperature modify the E. coli metabolic response, expression of chaperones, and structure of the IBs during rESAT-6 protein production in a thermoinducible system.

Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

BACKGROUND: Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. RESULTS: Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h-1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and ß-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. CONCLUSIONS: In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.