From shake flasks to bioreactors: survival of E. coli cells harboring pGST–hPTH through auto-induction by controlling initial content of yeast extract

Abstract

A high content of yeast extract in complex media can cause auto-induction of phage T7 RNA polymerase and the consequent expression of recombinant protein in Escherichia coli BL21(DE3) during long-term cultivation. Our study demonstrated that the auto-induction of recombinant protein varied in different vectors harboring heterologous genes. Trx, GST, and their fusion proteins such as GST-human parathyroid hormone (hPTH), expressed by pET32a (+), were easily auto-induced by media containing a high content of yeast extract; however, rtPA was not easily auto-induced when using pET22b (+), although both pET systems were under the control of T7lac promoter. Furthermore, the auto-induction of GST-hPTH may start within 1-2 h after inoculation in bioreactors, which is a deficiency in the scale-up from shake flasks to bioreactors. Our results indicated that too much yeast extract in bioreactor cultivations may be responsible for the early auto-induction of target proteins and consequent loss of cell viability and plasmid instability. To achieve a satisfactory yield, host cells with both high cell viability and plasmid stability were necessary for the starter cultures in shake flasks and pre-induction cultures in bioreactors. This could be achieved simply by controlling the initial content of yeast extract and its subsequent supplementation.

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

“…Despite being frequently used for protein production using T7-based E. coli expression systems in shake flask cultures [27,34], only a few investigations have considered bioreactor cultures based on auto-induction complex medium formulations [29,30,35]. …”

“…Cell viability and plasmid stability were evaluated by counting the final number of colonies formed [30]. …”

“…Biomass concentrations of up to 40 gDCW L -1 [29,30] can be obtained directly from batch experiments employing a mixture of glucose, glycerol, and lactose, with minimal requirements for handling of the expression culture [31]. However, this strategy has not yet been reported for PGA production in shake flask or bioreactor cultures of recombinant E. coli .…”

Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

“…Glycerol is included to (together with lactose) provide sustained growth during the induction phase (Studier 2005). In spite of the widespread use of auto-induction in shaker flask experiments, only few bioreactor cultures carried out with auto-induction media in batch (Giomarelli et al 2006; Blommel and Fox 2007a, b) or fed-batch mode (Jia et al 2011; Xu et al 2012) have been reported so far. In these studies, bioreactor media contain basically the same carbon and nitrogen sources, but the total carbon source concentration was increased by factors of approximately two (Blommel et al 2007a, b), three (Giomarelli et al 2006) or four (Xu et al 2012) in comparison to the sum of glucose, glycerol and lactose concentrations present in the original auto-induction ZYM-5052 complex formulation (Studier 2005).…”

“…In these studies, bioreactor media contain basically the same carbon and nitrogen sources, but the total carbon source concentration was increased by factors of approximately two (Blommel et al 2007a, b), three (Giomarelli et al 2006) or four (Xu et al 2012) in comparison to the sum of glucose, glycerol and lactose concentrations present in the original auto-induction ZYM-5052 complex formulation (Studier 2005). Consequently, maximum optical densities (600 nm) in the range of ~ 20 to 40 (Giomarelli et al 2006; Blommel et al 2007a, b; Jia et al 2011) were reported for batch experiments and of 140 for fed-batch cultures (Xu et al 2012). Nevertheless, none of these studies aimed to compare induction strategies.…”