Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems

Abstract

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH(4)(+) as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L(-1) product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-D-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.

Identification of bacterial laccase cueO mutation from the metagenome of chemical plant sludge

“…PCR products were ligated into the pET-30a(+) vector and transformed into BL21 (DE3) cells. The mutants were incubated in 96-well plates in an autoinduction medium at 30 °C with shaking at 200 rpm (Ukkonen et al 2013;Li et al 2011). After 24 h, the cells were harvested by centrifugation at 12,000g for 1 min, and the pellets were resuspended in 20 mM Tris/HCl buffer (pH 8.0) containing 20% sucrose, and the mixture was placed on ice for 30 min and centrifuged at 12,000g for 15 min.…”

Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems

“…If possible, autoinduction was used as a very convenient method for generating labeled proteins as it omits biomass monitoring for the correct timing of inducer addition and places the shift from growth to recombinant protein production under metabolic control of the expression host (Li et al 2011). Using autoinduction a mixture of glucose, glycerol and lactose is used as carbon substrate.…”

“…The composition and preparation of the defined labeling medium are based on the defined high cell density cultivation medium (Korz et al 1995) and the defined autoinduction medium (Li et al 2011). A general scheme for the usage and composition of the defined labeling medium including medium abbreviations are given in Table 1.…”

The kinetics of <i>Escherichia coli</i> B growth and bacteriophage T4 multiplication in SM-1 novel minimal culture medium

“…These media are developed with the aim to achieve the growth of microorganisms as well as a rich medium, or to enhance the activities of sev- Jia and Zhong (2011) have reported that the addition of Mg 2+ into a culture medium increases the production of anticancer ansamitocin P-3 (AP-3) by submerged cultures of Actinosynnema pretiosum. New-defined media can also be used to produce high-level recombinant protein using E. coli expression systems (Fu, 2010;Li et al, 2011). Synthetic minimal medium MCLMAN for Campylobacter jejuni strain NCTC 11168 is useful in physiological assays and responses to exogenous agents.…”