Optimized release of recombinant proteins by ultrasonication of E. coli cells.

The release kinetics of beta-galactosidase protein have been determined during small-scale ultrasonication of E. coli cells. Among several studied parameters, ionic strength and cell concentration have the least influence on the rate of protein recovery, whereas sample volume and acoustic power dramatically affect the final yield of soluble protein in the cell-free fraction. The analysis of these critical parameters has prompted us to propose a simple model for E. coli disintegration that only involves acoustic power and sample volume, and which allows prediction of optimal sonication times to recover significant amounts of both natural and recombinant proteins in a given set of relevant conditions.

[1]  M. Uhlén,et al.  Fusion proteins in biotechnology. , 1992, Current opinion in biotechnology.

[2]  A. Villaverde,et al.  An optimized ultrasonication protocol for bacterial cell disruption and recovery of β-galactosidase fusion proteins , 1994 .

[3]  A. Villaverde,et al.  Enhanced production of pL-controlled recombinant proteins and plasmid stability in Escherichia coli RecA+ strains. , 1993, Journal of biotechnology.

[4]  H. Blöcker,et al.  Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. , 1987, Gene.

[5]  H M Sassenfeld,et al.  Engineering proteins for purification. , 1990, Trends in biotechnology.

[6]  M. S. Doulah.  Mechanism of disintegration of biological cells in ultrasonic cavitation , 1977, Biotechnology and bioengineering.

[7]  A. Squartini,et al.  Experimental conditions may affect reproducibility of the beta-galactosidase assay. , 1992, FEMS Microbiology Letters.

[8]  A. Villaverde,et al.  Improved Mimicry of a Foot-and-Mouth Disease Virus Antigenic Site by a Viral Peptide Displayed on β-Galactosidase Surface , 1995, Bio/Technology.

[9]  A. Squartini,et al.  Experimental conditions may affect reproducibility of the β-galactosidase assay , 1992 .

[10]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[11]  A. Middelberg,et al.  A Correlation for the Effective Strength of Escherichia coli during Homogenization , 1993, Biotechnology progress.

[12]  B. Bachmann,et al.  Pedigrees of some mutant strains of Escherichia coli K-12. , 1972, Bacteriological reviews.

[13]  J. Corchero,et al.  The position of the heterologous domain can influence the solubility and proteolysis of beta-galactosidase fusion proteins in E. coli. , 1996, Journal of biotechnology.

[14]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[15]  H. Umakoshi,et al.  Optimal disruption methods for the selective recovery of β-galactosidase from Escherichia coli , 1995 .

[16]  A. Blechl,et al.  Purification and characterization of wheat α-gliadin synthesized in the yeast, Saccharomyces cerevisiae , 1992 .

[17]  C. Cooney,et al.  Fermentation and Enzyme Technology , 1979 .

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  W. Coakley,et al.  Kinetics of protein release from yeast sonicated in batch and flow systems at 20 kHz , 1972 .

[20]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .