A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants.
A novel gene fusion system to express and purify small recombinant proteins in Escherichia coli has been constructed. The concept allows for affinity purification of soluble gene products by sequential albumin- and Zn2(+)-affinity chromatography. The dual-affinity system is well suited for expression of unstable proteins as only full-length protein is obtained after purification and proteins gain proteolytic stability in the fusion protein. Here we show that the dual-affinity approach can be used for the expression of various unstable derivatives of a single IgG-binding domain based on staphylococcal protein A. Analysis of the proteolytic stabilities and the IgG-binding properties of the different mutant proteins suggest that the model for the structure of an IgG-binding domain must be re-evaluated.
Royal Institute of Technology, Department of Biochemistry and Biotechnology, Stockholm, Sweden.
B
B
Jansson
C
C
Palmcrantz
M
M
Uhlén
B
B
Nilsson
D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012709:Serum Albumin / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000235:genetics*; D015032:Zinc / Q000378:metabolism
D004269:DNA, Bacterial; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D012709:Serum Albumin; D013205:Staphylococcal Protein A; D015032:Zinc