Dialysis strategies for protein refolding: preparative streptavidin production.

We have investigated different dialysis strategies for the refolding of recombinant streptavidin, and present a novel dialysis setup featuring gradual dilution dialysis and continuous protein feeding into a dialysis sack. A denaturing dialysis buffer is exchanged gradually by dilution with refolding buffer and it is demonstrated that the refolding yield can be increased from 45 to 75% by lowering the dilution rate. In addition, continuous feeding of protein to the dialysis sack increases the yield by 5 to 10%. The principle of gradual dilution dialysis is amenable to stringent regulation and we suggest it to be applied for other insoluble protein targets.

[1]  A. Villaverde,et al.  Construction and deconstruction of bacterial inclusion bodies. , 2002, Journal of biotechnology.

[2]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[3]  M. Karp,et al.  Identification of biotinylated molecules using a baculovirus-expressed luciferase-streptavidin fusion protein. , 1996, BioTechniques.

[4]  A. Middelberg,et al.  Preparative protein refolding. , 2002, Trends in biotechnology.

[5]  C. Cantor,et al.  Expression of a cloned streptavidin gene in Escherichia coli. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. Chilkoti,et al.  Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A. Sidoli,et al.  Production of a soluble and functional recombinant streptavidin in Escherichia coli. , 1998, Protein expression and purification.

[8]  R. Rudolph,et al.  In vitro folding of inclusion body proteins , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  L. Thompson,et al.  Construction and expression of a synthetic streptavidin-encoding gene in Escherichia coli. , 1993, Gene.

[10]  V. Nagarajan,et al.  Secretion of streptavidin from Bacillus subtilis , 1993, Applied and environmental microbiology.

[11]  P. Goodenough,et al.  A novel sequential procedure to enhance the renaturation of recombinant protein from Escherichia coli inclusion bodies. , 1992, Protein engineering.

[12]  Johannes Buchner,et al.  Protein Aggregation in vitro and in vivo: A Quantitative Model of the Kinetic Competition between Folding and Aggregation , 1991, Bio/Technology.

[13]  Guifeng Zhang,et al.  Dual gradient ion-exchange chromatography improved refolding yield of lysozyme. , 2002, Journal of chromatography. A.

[14]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.