Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display.
The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.
Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
Nina
N
Kronqvist
John
J
Löfblom
Denise
D
Severa
Stefan
S
Ståhl
Henrik
H
Wernérus
D000086042:3C Viral Proteases; D015374:Biosensing Techniques; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013210:Staphylococcus / Q000235:genetics*; D014169:Transformation, Bacterial; D014764:Viral Proteins / Q000378:metabolism*
D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014764:Viral Proteins; C515865:Z(HER2.4)2 affibody; C508053:ERBB2 protein, human