Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides.
An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.
Huang
Xu
X
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Zhang
Xian-En
XE
Zhou
Ya-Feng
YF
Zhang
Zhi-Ping
ZP
Cass
Anthony E G
AE
D000345:Affinity Labels; D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D008024:Ligands; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000345:Affinity Labels; D008024:Ligands; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase