Mutant proteins of human lipocalin 2 (lcn2, hngal) with affinity for a given target
A mutated protein and target technology, which is applied to human lipocalin 2 (Lcn2) with affinity for a given target, which can solve the problem of easy oligomerization of L19 scFv fragments
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Embodiment 1
[0190] Example 1: Construction of a mutant Lcn2 phage display library
[0191] A combinatorial library of Lcn2 variants was generated based on cloned cDNA (Breustedt et al. (2006) Biochim. Biophys. Acta 1764, 161-173) carrying the amino acid substitution Cys87Ser to remove a single unpaired thiol side chain (Goetz et al. (2000) Biochemistry 39, 1935-1941 ) and Gln28His to introduce a second BstXI restriction site. Mutations in this region and polymerase Chain reaction (PCR) assembly, at this time using such as in figure 1 One-pot amplification reaction of oligodeoxynucleotides (SEQ ID NO: 1-10). Oligodeoxynucleotides are designed such that primers having SEQ ID NO: 1-4 correspond to the coding strand and are at amino acid positions 36, 40, 41, 49, 52 or 68, 70, 72, 73, 77, 79, 81 or 96, respectively , 100, 103, 106 or 125, 127, 132, 134 carried degenerate codons, while primers with SEQ ID NO: 5-8 corresponded to the non-coding strand and carried no degenerate codons or a...
Embodiment 2
[0197] Example 2: Preparation of different forms of Aβ targets
[0198] The Aβ40 peptide (SEQ ID NO: 29) corresponding to amino acids 1 to 40 of the mature β-amyloid sequence (Dodel et al. (2003) Lancet Neurology 2, 215-220) was purchased as a synthetic lyophilized peptide with Lysine spacer-linked C-terminal biotin groups (Peptide Speciality Laboratory, Heidelberg, Germany) or in unlabeled form (W.M. Keck Laboratory, New Haven, USA). Obtained by dissolving up to 5 mg of the peptide in 0.5 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma-Aldrich, Steinheim, Germany) for at least 0.5 h at room temperature A homogeneous monomeric Aβ40 was obtained. Afterwards, HFIP was evaporated in a SpeedVac concentrator, and Aβ40 was dissolved in an appropriate volume of distilled water, followed by sonication (Bandelin, Sonorex, RK100, Germany) in cold water for 15 minutes. After filtration with a 0.22 μm filter (Spin-X centrifuge tube filter; Corning, USA), by using the calculated e...
Embodiment 3
[0207] Example 3: Selection of Lcn2 muteins with affinity for Aβ40 peptide by phage display
[0208] For each panning cycle, 2% (w / v) in PBS / T (PBS containing 0.1% (v / v) Tween20 [polyoxyethylene sorbitan monolaurate; AppliChem, Darmstadt, Germany]) ) BSA blocked PBS (4mMKH 2 PO 4 ,16mMNa 2 HPO 4 , 115mMNaCl, pH7.4) about 10 12 A recombinant phagemid for 1 hour. 50 μL and 25 μL of multiple streptavidin-coated magnetic bead suspensions (Dynabeads M-280 Streptavidin; DynalBiotech, Invitrogen, Karlsruhe, Germany and Streptavidin Magnetic Particles; Roche Diagnostics, Mannheim, Germany) were washed with PBS / T, respectively, and then washed with PBS / T Block with 2% (w / v) BSA in T for 1 hr. 25 μL of blocked beads were used to pre-adsorb blocked phagemids to remove phagemids specific to the column, 50 μL was used afterwards for the first selection cycle.
[0209] Blocked phagemids were incubated with 25 μL of washed and blocked streptavidin-coated magnetic beads. The beads w...
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