Residue:Article Title: CD40L binding polypeptide
Article Snippet: The overlap PCR was performed under the following conditions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. In addition, an RD-05 mutant antibody [RD-05 (L115E)] wherein leucine at residue 115 of the heavy-chain constant region of human IgG4 was substituted with glutamic acid (L115E) was produced in the following manner. .. Specifically, a gene comprising the heavy-chain variable region and proximal heavy-chain constant region including overlapping 11 amino acids encompassing residue 115 of the human IgG4 constant region was amplified by PCR, and the remaining distal heavy-chain constant region including overlapping 11 amino acids encompassing residue 115 was amplified by PCR, followed by overlap PCR The PCR amplification reactions were performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTGTGGCTGTTTACA-3′ (SEQ ID NO:25); human IgG4 constant 115 region CH reverse primer: 5′-CAGGAACACGGAAGGTCCGCCTTCAAATTCAGGGGCAGG-3′ (SEQ ID NO:46); human IgG4 constant 115 region CH forward primer: 5′-CCCTGCCCTGCCCCTGAATTTGAAGGCGGACCTTCCGTG-3′ (SEQ ID NO:47); and human IgG4 constant region CH reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43). .. The PCR amplification reactions were performed under the following reactions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. for 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. The overlap PCR was performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTTGTGGCTGTTTACA-3′ (SEQ ID NO:25); human IgG4 constant region CH reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43).
Amplification:Article Title: CD40L binding polypeptide
Article Snippet: The overlap PCR was performed under the following conditions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. In addition, an RD-05 mutant antibody [RD-05 (L115E)] wherein leucine at residue 115 of the heavy-chain constant region of human IgG4 was substituted with glutamic acid (L115E) was produced in the following manner. .. Specifically, a gene comprising the heavy-chain variable region and proximal heavy-chain constant region including overlapping 11 amino acids encompassing residue 115 of the human IgG4 constant region was amplified by PCR, and the remaining distal heavy-chain constant region including overlapping 11 amino acids encompassing residue 115 was amplified by PCR, followed by overlap PCR The PCR amplification reactions were performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTGTGGCTGTTTACA-3′ (SEQ ID NO:25); human IgG4 constant 115 region CH reverse primer: 5′-CAGGAACACGGAAGGTCCGCCTTCAAATTCAGGGGCAGG-3′ (SEQ ID NO:46); human IgG4 constant 115 region CH forward primer: 5′-CCCTGCCCTGCCCCTGAATTTGAAGGCGGACCTTCCGTG-3′ (SEQ ID NO:47); and human IgG4 constant region CH reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43). .. The PCR amplification reactions were performed under the following reactions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. for 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. The overlap PCR was performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTTGTGGCTGTTTACA-3′ (SEQ ID NO:25); human IgG4 constant region CH reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43).
Polymerase Chain Reaction:Article Title: CD40L binding polypeptide
Article Snippet: The overlap PCR was performed under the following conditions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. In addition, an RD-05 mutant antibody [RD-05 (L115E)] wherein leucine at residue 115 of the heavy-chain constant region of human IgG4 was substituted with glutamic acid (L115E) was produced in the following manner. .. Specifically, a gene comprising the heavy-chain variable region and proximal heavy-chain constant region including overlapping 11 amino acids encompassing residue 115 of the human IgG4 constant region was amplified by PCR, and the remaining distal heavy-chain constant region including overlapping 11 amino acids encompassing residue 115 was amplified by PCR, followed by overlap PCR The PCR amplification reactions were performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTGTGGCTGTTTACA-3′ (SEQ ID NO:25); human IgG4 constant 115 region CH reverse primer: 5′-CAGGAACACGGAAGGTCCGCCTTCAAATTCAGGGGCAGG-3′ (SEQ ID NO:46); human IgG4 constant 115 region CH forward primer: 5′-CCCTGCCCTGCCCCTGAATTTGAAGGCGGACCTTCCGTG-3′ (SEQ ID NO:47); and human IgG4 constant region CH reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43). .. The PCR amplification reactions were performed under the following reactions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. for 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. The overlap PCR was performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTTGTGGCTGTTTACA-3′ (SEQ ID NO:25); human IgG4 constant region CH reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43).
Article Title: CD40L binding polypeptide
Article Snippet: .. The PCR was performed under the following conditions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. for 45 sec, 52° C. for 45 sec, and 72° C. for 1 min; 72° C. for 7 min; stop at 4° C. Overlap PCR for fusing the heavy-chain variable region and the human IgG4 constant region, and overlap PCR for fusing the light-chain variable region and the human light-chain kappa region, were performed using the following primers: RD-05 VH region forward primer: 5′-CTCGAGATGAGGGTGTTAATTCTTTTGTGGCTGTTTACA-3′ (SEQ ID NO: 25); human IgG4 constant region CH region reverse primer: 5′-GAATTCTCATTTGCCCAGGCTCAGGGACAGGGACTTCTG-3′ (SEQ ID NO:43); RD-05 VL region forward primer: 5′-CTCGAGATGGAGACAGATACACTTTTGCTATGGGTTCTG-3′ (SEQ ID NO:29); human light-chain kappa CL region reverse primer: 5′-GAATTCTCAACATTCGCCCCTGTTAAAGCTTTTTGTGAC-3′ (SEQ ID NO:45). .. The overlap PCR was performed under the following conditions: 94° C. for 4 min; 35 cycles, each consisting of 94° C. 1 min, 61° C. for 1 min, and 72° C. for 1 min; 72° C. for 5 min; stop at 4° C. In addition, an RD-05 mutant antibody [RD-05 (L115E)] wherein leucine at residue 115 of the heavy-chain constant region of human IgG4 was substituted with glutamic acid (L115E) was produced in the following manner.
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