A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products.
An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag Z(SPA-1), enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag Z(SPA-1), was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (P(T7)) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His(6)) tag in frame with the Z(SPA-1) tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.
Division of Molecular Biotechnology, Department of Biotechnology, Royal Institute of Technology (KTH), SCFAB, SE-10691 Stockholm, Sweden.
Susanne
S
Gräslund
Malin
M
Eklund
Ronny
R
Falk
Mathias
M
Uhlén
Per-Ake
PA
Nygren
Stefan
S
Ståhl
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D007093:Imidazoles; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics*; D013205:Staphylococcal Protein A; D013737:Testis / Q000737:chemistry
D018076:DNA, Complementary; D007093:Imidazoles; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C005954:imidazoleacetic acid