Advanced Journal of Microbiology Research

ISSN 2736-1756

Advanced Journal of Microbiology Research Vol. 2004

Available online at http://internationalscholarsjournals.org/journal/ajmr

© 2004 International Scholars Journals

Full Length Research Paper

Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-α, under different growth conditions

Thereza Christina Vessoni Penna*1, Marina Ishii1, Luciana Cambricoli de Souza 1, Olivia Cholewa2

1Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, SP, Brazil.

2Molecular Probes, Inc. Eugene, Or, USA. 97402. Email: [email protected].

Accepted 25 November 2003

Abstract

The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (24-1) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4oC prior to incubation at 37oC; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl–β-D-thiogalactopyranoside) and (iv) the addition of IPTG at set cell densities (OD660 0.01-0.8). GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column. The cultures grown at 37oC/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4oC/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG and (iv) IPTG addition at OD660~0.01. On the other hand, at 37oC/ 8 h, GFPuv expression was dependent upon agitation of broth cultures at 200 rpm and the IPTG addition at the beginning of the growth exponential phase.

Key words: Green fluorescent protein (GFPuv), Escherichia coli DH5-α, growth kinetic parameters, expressed GFPuv kinetic parameters, three phase partitioning extraction (TPP).