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Considering the T m values and binding affinities of all PV2 and PV3 mutants, PV2-S26pAcF, PV2-S71PAcF, PV3-S85pAcF and PV3-N77pAcF which showed significantly improved stability and binding affinity, were selected for 2 times 2 combination, resulting in four pairs of BsNbs