Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries.
mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.
School of Pharmacy and Carolina Center for Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Bao-Cheng
BC
Huang
Rihe
R
Liu
D020816:Amino Acid Motifs; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D012333:RNA, Messenger