Study on peptide-peptide interaction using high-performance affinity chromatography and quartz crystal microbalance biosensor

Abstract

The specific interaction between sense and antisense peptides was studied by high-performance affinity chromatography (HPAC) and quartz crystal microbalance (QCM) biosensor. Fragment 1-14 of human interferon-β (hIFN-β) was chosen as sense peptide and its three antisense peptides (AS-IFN 1, AS-IFN 2, and AS-IFN 3) were designed according to the degeneracy of genetic codes. The affinity column was prepared with sense peptide as ligand and the affinity chromatographic behavior was evaluated. Glu-substituted antisense peptide (AS-IFN 3) showed the strongest binding to immobilized sense peptide at pH 7.5. A quartz crystal microbalance-flow injection analysis (QCM-FIA) system was introduced to investigate the recognition process in real-time. The equilibrium dissociation constants between sense peptide and AS-IFN 1, AS-IFN 2 and AS-IFN 3 measured 2.08×10 −4 , 1.31×10 −4 and 2.22× 10 −5 mol/L, respectively. The mechanism study indicated that the specific recognition between sense peptide and AS-IFN 3 was due to sequence-dependent and multi-modal affinity interaction.antisense peptide, degeneracy, high-performance affinity chromatography, quartz crystal microbalance biosensor, human interferon-β Antisense peptides are the analogical equivalent of antisense oligonucleotides. By definition, a sense peptide is coded by the nucleotide sequence of the sense (positive) strand of DNA. Conversely, an antisense peptide is coded by the nucleotide sequence of the antisense (negative) strand of DNA. Usually the antisense strand of DNA cannot be translated in almost all organisms, but they can be synthesized chemically. As predicted by Mekler [1] and first shown experimentally by Bost et al. [2] , an antisense peptide could interact with the corresponding sense peptide at some degree of selectivity. The interaction between sense and antisense peptides has been reported [3,4] . As such interaction is specific and selective, its application in affinity technology has been widely achieved. For example, antisense peptide-immobilized chromatographic supports have been employed to isolate native proteins like recombinant c-raf protein [5] , the Arg 8 -vasopressin-receptor complex [6] and human interferon-β [7] . Moreover, Davids et al. [8] developed antisense peptides as the selective inhibitors of cytokine interleukin I in 1997. Since then, antisense peptides have been designed and selected as inhibitors of cytokine interleukin-1β [9] , interleukin 18 [10] and β-amyloid peptide 1-40 [11] . In our previous work, the inhibitor of influenza A virus was designed and screened out using antisense peptide based combinatorial peptide libraries [12] .Till now, the mechanism of interactions between sense and antisense peptides has not been clarified. Two main hypotheses have been put forward to explain the specific interactions between sense peptide and antisense peptide. As defined by Heal et al. in 2002 [4] , one

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