Question

3) Suppose that structural analysis of adalimumab binding to its target tumor necrosis factor suggests that hanging one amino acid in the light chain variable region from Asp to Glu will enhance binding activity. Describe in conceptual detail how you will create a mutation that changes the codon GAC to GAG in the light chain coding sequence.

please give a detailed step by step laboratory procedure. thank you.

Answer 1

Q. Suppose that structural analysis of adalimumab binding to its target tumor necrosis factor suggests that hanging one amino acid in the light chain variable region from Asp To Glu will enhance binding activity. Describe in conceptual detail how you will create a mutation that changes the codon GAC to GAG in the light chain coding sequence.

Ans. Adalimumab was cloned, expressed and purified subsequent reported events. EcoRV and XbaI position were additional to the 5′-end of the heavy chain variable section gene (V_H), and an NheI position was added to the 3′-end. The PCR produce cloned into the pGEM-T vector and the sequence of the invention was established throughout DNA sequencing. V_H was expunged throughout EcoRV and NheI digestion and then introduced into the EcoRV/NheI position of the pAH4604 vector including the human gamma-1 stable region gene (C_H). The consequential pAH4604-V_H vector was cut with XbaI and BamHI. The 3.3-kb portion including the human antibody heavy chain gene was cloned into the pcDNA3.1(−) vector which is digested through the similar restriction enzymes and give in the heavy chain expression vector pcDNA3.1(−)V_HC_H. The human κ chain stable cDNA is achieved as a 0.3-kb PCR product consequent from pAG4622. The light chain variable region gene (V_L) of Adalimumab is merged to the 5′-end of C_L throughout the partly cover PCR technique. The consequential human light chain gene (V_LC_L), which has a Hind III position upstream of the start codon and an EcoRI position downstream of the stop codon, is cloned into the pGEM-T vector. The sequence of the chain is verified. V_LC_L is expurgate throughout Hind III and EcoRI digestion and ligated into pcDNA3.1. The light and heavy chain appearance vector is co-transfected into Chinese hamster ovary K1 cells with Lipofectamine 2000.

The Fab portion of Adalimumab for the crystallographic investigation is achieved during papain digestion of the antibody. The digested protein section is laden onto a Protein A-Sepharose 4 FF column. The Fab portion eluted in the flood during alienated from the Fc fragment and further purified through ion exchange chromatography using a Q-Sepharose FF column. The protein sample is concentrated to ∼10 mg/ml and after that replaces to a reserve buffer including 10 mm Tris-HCl (pH 8.0) and 100 mm NaCl. TNFα was subsequently assorted through surplus of Adalimumab Fab, and the complex is purified during gel filtration chromatography. This complex is dialyzed beside 50 mm Tris-HCl (pH 8.0) and 150 mm NaCl and concentrated to 30 mg/ml. superimpose TNFα in the TNFα-Adalimumab Fab complex among free form capitulate a root mean square deviation of 0.9 Å intended for every one of the Cα atoms.

The Adalimumab Fab present canonical β-sandwich immunoglobulin fold among the heavy chain folding into the V_H and C_H domains and the light chain folding into the V_L and C_L domains. The light chain of Adalimumab cooperate among strands A and C, as well as the A-A′ and the E-F loops of TNFα. Remains _AdaAsp-1 and _AdaArg-93 of CDR L3 structure, three hydrogen bonds and a salt bridge with _TNFPro-20, _TNFGln-21, and _TNFGlu-23 at the commencement of strand A and the A-A′ loop of TNFα. In general system of intermolecular side chain hydrogen bonds among CDR L1 and strand C of TNFα donate the most of the light chain interface and site of the side chain of _AdaArg-30, _AdaAsn-31, and _AdaTyr-32 of CDR L1 cooperating among _TNFLys-65 and _TNFGln-67 of TNFα. The crossing point among the heavy chain of Adalimumab and TNFα is chiefly collected of the remains in the G-H loop, numerous amino acids in strand D, and the D-E loop of an neighboring TNFα protomer. _AdaHis-57 in CDR H2 and _AdaSer-103 and _AdaThr-104 in CDR H3 create hydrogen bonds among _TNFAla-145 to _TNFSer-147 in the G-H loop.