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Abstract

IDENTIFICATION OF ANTIBODY TARGETS USING PHOTOACTIVATED CROSS-LINKERS LOCATED IN THE BINDING SITE

*Iyam Solomon Offem, Oko Gregory Elayeche and Okoi Paul Emmanuel

ABSTRACT

We converted the monoclonal antibody (STRO-1), into a recombinant antibody fragment and subsequent alteration of the complementarity determining region (CDR 3) of the variable region of the heavy chain (VH) by site-directed mutagenesis (SDM). We modified the nucleotide codon (TAT) which encode the amino acid tyrosine (Y), to an amber codon (TAG) in DNA or (UAG) in mRNA in the CDR3 of the heavy chain variable region (VH) and was transformed into E.coli (TOP10, XL1-Blue) cells. The amino acid sequence of both the heavy and light chains obtained from US patent no: 8,101,155 B2 (Simmons & Torok- Storb, 1991) were translated to the corresponding nucleotide sequence. The variable region of the heavy chain (VH) and light chain (VL) were amplified by PCR after 30 cycles reaction. However, we were completely unable to clone the native STRO-1 genes that encode for the VH and VL fragments into the pHenIX cloning vector after several attempts. The DNA fragment which contains the sequence that encode the (VH) gene was then cloned into a TA cloning vector (pCR 2.1, 3.9kb) after PCR amplification of the altered target of interest. Following ligation reaction, we transformed E.coli (TOP10) cells, grown overnight, purified, and sequenced for determination of the incorporation of the modified nucleotide sequence. Comparison of the mutated CDR3 of the variable heavy (VH) chain with the native sequence after sequencing was carried out showed that mutation has not occurred at the desired positions due to mismatch of some codons possibly due to incorrect primer design.

Keywords: Strol-1, monoclonal antibodies, site directed mutagenesis, sequencing, cloning, expression vectors, recombinant fragment.


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