Immunoglobulin binding proteins for affinity purification
A binding protein, affinity technology, applied in the field of immunoglobulin binding protein, can solve the problem of losing the binding ability of immunoglobulin
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[0067] The present invention will now be further described. Different embodiments of the invention are defined in more detail in the following paragraphs. Each embodiment defined below can be combined with any other embodiment unless expressly stated to the contrary. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or feature indicated as preferred or advantageous.
[0068] In one embodiment, the Ig protein comprises one or more domains, wherein at least one domain comprises the amino acid sequence of any one of SEQ ID NO: 1-10, or the amino acid of any one of SEQ ID NO: 1-7 sequence, or at least 89.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto or consist essentially of or consist of amino acids of . In some embodiments, the Ig protein comprises one or more domains, wherein at least one domain comprises...
Embodiment 1
[0125] Example 1: Production of Ig binding proteins of the invention
[0126] Artificial Ig-binding ligands were originally generated by a process of shuffling naturally occurring protein A domains and protein A domain variants (eg, Z domains). In more detail, a shuffling process as understood herein is a process resulting in the assembly of an artificial amino acid sequence starting from a different set of known amino acid sequences. The shuffling process comprises the following steps: a) providing the sequences of the five naturally occurring Protein A domains E, B, D, A and C and the Protein A variant domain Z; b) alignment of said sequences; In silico (insilico) statistical fragmentation to identify recombined subsequences and then d) assembly of new artificial sequences of the various fragments to generate mosaic products, ie new amino acid sequences. The fragments generated in step c) have any length, for example, if the length of the fragmented parent sequence is n, th...
Embodiment 2
[0129] Example 2: Expression of Ig binding proteins
[0130] BL21(DE3) competent cells were transformed with an expression plasmid encoding an Ig-binding protein. Cells were plated on selective agar plates (kanamycin) and incubated overnight at 37°C. Inoculate the preculture from a single colony into 50 ml 2xYT medium supplemented with 50 μg / ml kanamycin and incubate at 37 °C for 17 h at 200 rpm in a 500 mL Erlenmeyer flask in a conventional orbital shaker. OD 600 The reading should be in the range of 4-6. In a 1 L thick-walled Erlenmeyer flask, in 300 ml ultra-rich medium (2% glucose, 5% yeast extract, 0.89% glycerol) supplemented with 50 μg / ml kanamycin and trace elements (see Studier 2005) , 0.76% lactose, 250mM MOPS, 202mM TRIS, 10mM MgSO 4 , pH 7.4, modified H15 medium composed of antifoam Se15), using an adjusted initial OD of 0.3 600 , inoculate the main culture from a previous overnight culture. Transfer the culture to a resonant acoustic mixer (RAM bio ) and in...
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