title: Enhancing thermo-induced recombinant protein production in Escherichia coli by temperature oscillations and post-induction nutrient feeding strategies.
abstract: Traditional strategies for production of thermo-induced recombinant protein in Escherichia coli consist of a two-phase culture, with an initial growth stage at low temperature (commonly 30°C) followed by a production stage where temperature is increased stepwise (commonly up to 42°C). A disadvantage of such strategies is that growth is inhibited upon temperature increase, limiting the duration of the production stage and consequently limiting recombinant protein production. In this work, a novel oscillatory thermo-induction strategy, consisting on temperature fluctuations between 37 and 42°C or 30 and 42°C, was tested for improving recombinant protein production. In addition, the induction schemes were combined with one of three different nutrient feeding strategies: two exponential and one linear. Recombinant human preproinsulin (HPPI), produced under control of the λP(L)-cI857 system in the E. coli BL21 strain, was used as the model protein. Compared to the conventional induction scheme at constant temperature (42°C), longer productive times were attained under oscillatory induction, which resulted in a 1.3- to 1.7-fold increase in maximum HPPI concentration. Temperature oscillations led to a 2.3- to 4.0-fold increase in biomass accumulation and a decrease of 48-62% in the concentration of organic acids, compared to conventional induction. Under constant induction, growth ceased upon temperature increase and the maximum concentration of HPPI was 3.9 g/L, regardless of the post-induction feeding strategy used. In comparison, the combination of temperature oscillations and a high nutrient-feeding rate allowed sustained growth after induction and reaching up to 5.8 g/L of HPPI.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, CP 62210 Cuernavaca, Morelos, Mexico.
authors.forename: Luis
authors.initials: L
authors.lastname: Caspeta
authors.forename: Alvaro R
authors.initials: AR
authors.lastname: Lara
authors.forename: Néstor O
authors.initials: NO
authors.lastname: Pérez
authors.forename: Noemí
authors.initials: N
authors.lastname: Flores
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolívar
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
mesh_terms: D018533:Biomass; D019149:Bioreactors; D004926:Escherichia coli / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000096:biosynthesis*; D011498:Protein Precursors / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature
chemical_list: D007328:Insulin; D011498:Protein Precursors; D011994:Recombinant Proteins; C013737:preproinsulin; D005947:Glucose
keywords: Bacteriophage lambda P(L) promoter; E. coli; Recombinant protein; Temperature induction; Temperature oscillations