A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
2002 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, no 1, 41-50 p.Article in journal (Refereed) Published
An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-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 ZSPA-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 (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-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.
Place, publisher, year, edition, pages
2002. Vol. 99, no 1, 41-50 p.
Affibody; Affinity blotting; Affinity chromatography; cDNA; Combinatorial protein engineering; Gene expression; Proteomics; Staphylococcus aureus protein A
Immunology in the medical area
IdentifiersURN: urn:nbn:se:kth:diva-5913ISI: 000178484900003OAI: oai:DiVA.org:kth-5913DiVA: diva2:10447
QC 201008202006-06-022006-06-022010-08-24Bibliographically approved