HOW TO AVOID BACTERIAL INCLUSION BODIES? Ana Sofia Pinaa,b*, Luciana Tiemi Caraça, Christopher R. Loweb and A.

Cecília A. Roquea
a

REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK * asp14526@campus.fct.unl.pt The production of recombinant proteins in Escherichia coli presents several advantages, such as simplicity, well established methods for genetic manipulation, high products yields, rapid expression and cost effectiveness [1]. However, a major drawback is that protein expression can lead to the formation of insoluble aggregates. These aggregates, inclusion bodies (IBs), are formed by unfolded or highly misfolded polypeptides [2]. In order to obtain the required protein in a soluble and functional format, IBs usually undergo solubilization and refolding processes, which are expensive, time consuming and highly proteindependent [2]. Different alternatives have been studied to avoid IBs formation. The culture conditions, temperature and inducing agents levels (Isopropyl β-D1-thiogalactopyranoside, IPTG), are parameters that can be easily modified to minimize IBs formation. The main aim of this work was to study the impact of different temperatures after induction (27, 30, 33 and 37ºC) and IPTG levels (0.1, 0.5 and 1 mM) on the overall productivity of Green Fluorescent Protein (GFP) fusion proteins and on the ratio between soluble GFP fusion protein and IB formation. The fusion protein used on these studies was a GFP fused at the N – terminal to a hydrophobic peptide. As a model system, E. coli BL21(DE3) with pET 21C plasmid containing the gene encoding for the GFP fusion protein have been used. Protein expression was monitored through fluorescence intensity on a high-throughput format and SDS-PAGE 50000 45000 analysis. Optical density measurements 40000 were also performed. The results showed 35000 that the productivity of the GFP fusion 30000 25000 proteins increases as the level of inducing 20000 agent increases, being significantly 15000 10000 improved when using 1mM of IPTG at 5000 lower temperatures. For these conditions, 0 IBs formation was minimized and the 27⁰C 30⁰C 33⁰C 37⁰C amount of soluble protein produced Temperature improved by a factor of 10 (Fig. 1).
Fig. 1 – Fluorescence intensity of the soluble protein fraction produced at different growth temperatures and 1mM IPTG concentration.

1. 2.

Demain, A.L. and P. Vaishnav, Biotechnology Advances, 2009. 27(3): p. 297-306. García-Fruitós, E., et al., Trends in Biotechnology, 2012. 30(2): p. 65-70.

fluorescence intensity (AU)

Sign up to vote on this title
UsefulNot useful

Master Your Semester with Scribd & The New York Times

Special offer for students: Only $4.99/month.

Master Your Semester with a Special Offer from Scribd & The New York Times

Cancel anytime.