Fusion protein comprising ccl3 variant and use thereof cross-reference to related applications
A fusion protein and variant technology, applied to medical preparations containing active ingredients, fusion polypeptides, peptide/protein components, etc., can solve the problems of reduced affinity and increased residence time, and achieve low risk of immunogenicity, Improve the effect of persistence in the body
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preparation Embodiment 1
[0093] Preparation Example 1. Preparation and purification of fusion proteins comprising CCL3 variants
[0094] Mutation studies were performed on CCL3 to improve the physical properties and activity distribution of CCL3 in the CCL3-Fc structure.
[0095] Specifically, in order to determine whether the difference in activity between the α-type (hereinafter referred to as CCL3α) and β-type (hereinafter referred to as CCL3β) of CCL3 protein is maintained even after Fc fusion, various variants were constructed. In addition, in order to inhibit the precipitation of CCL3 in the Fc fusion protein type, a variant in which aspartic acid at position 27 was replaced by alanine was designed.
[0096] The location, sequence information, target and expected effect of each mutation introduced into CCL3 are summarized in Table 1 below.
[0097] [Table 1]
[0098]
[0099] Amino acids were encoded in the expression vector to express the three constituent elements in the order of CCL3 var...
preparation Embodiment 1-2
[0104] Preparation Example 1-2. Preparation of plasmid DNA for expressing CCL3 variant fusion protein
[0105] By transforming each expression vector prepared in Preparation Example 1-1 into Escherichia coli, a large amount of plasmid DNA for expression was obtained. Each expression vector was transduced into E. coli having a weakened cell wall by heat shock, and the resulting transduced E. coli were spread on LB plates to obtain colonies. The obtained colonies were inoculated into LB medium and cultured at 37° C. for 16 hours, and 100 mL of Escherichia coli having the corresponding expression vectors in the cells were respectively obtained. After removal of the medium by centrifugation, solutions P1, P2, and P3 (QIAGEN, Cat. No. 12963) were added to E. coli to obtain a DNA turbid solution by disrupting the cell wall and separating proteins and DNA. Plasmid DNA was purified from the obtained DNA cloudy solution by using a Qiagen DNA purification column. The eluted plasmid DN...
preparation Embodiment 1-3
[0106] Preparation Example 1-3. Expression of fusion protein in CAP-T cells
[0107] Human cell lines were transformed with each of the plasmid DNAs isolated in Preparation Example 1-2. Each plasmid DNA was transduced into CAP-T cells (CEVEC) simultaneously cultured in PEM medium (Life technologies) by using PEI solution (Polyplus, catalog number 101-10N). The mixed solution of DNA and PEI solution was mixed with the suspended cells by using Freestyle 293 expression medium from Invitrogen Corporation, which was cultured at 37°C for 5 hours, and then PEM medium was added. After culturing at 37°C for 5 to 7 days, the cells were removed by centrifugation to obtain a supernatant containing the CCL3 variant fusion protein.
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