Recombinant Fusion Proteins and Libraries from Immune Cell Repertoires
a fusion protein and immune cell technology, applied in the field of protein engineering, can solve the problems of expensive and laborious antibody drug discovery process, and the artificial generation of a representative immune repertoire from an individual with cognate paired heavy and light chain immunoglobulin or t cell receptors has not been achieved
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example 1
scFv Library Generation
[0158]Methods and compositions of the invention will now be discussed relative to scFv library generation.
[0159]Preparation of Beads
[0160]2× LiCl buffer was prepared as follows: For 250 mL of 2× LiCl buffer, 10 mL of 1M Tris (pH 7.5), 31.25 mL 8M LiCl, and 1 mL of 500 mM EDTA were added to 180.25 mL molecular grade water. 2× lysis buffer was prepared as follows: For each 1 mL of 2× lysis buffer, 890 μL of 2× LiCl stock, 90 μL of molecular grade water, 10 ul of 1M DTT, and 10 μL of Tween 20 were mixed to produce 1 mL solution of 2× lysis buffer.
[0161]Biotinylated IgK and IgG probes, which bind to the IgK and IgG constant regions, respectively, were synthesized with the following sequences: SEQ ID NO: 6-7. Probes were each added to 100 μL of 2× lysis buffer to a final concentration of 10 μM.
[0162]A 1 μM solution of streptavidin magnetic beads from New England Biolabs (NEB 514205) was gently rocked at room temperature for 30 minutes. 200 μL of the streptavidin ma...
example 2
Antibody Generation
[0177]Generation of Expression Constructs
[0178]An scFv library (including, e.g., SEQ ID NO: 1) was linearized by PCR using standard PCR methods and forward and reverse primers (e.g., SEQ ID NO: 4,5). An insert containing a second AOX1 promoter was synthesized by a gene synthesis vendor (IDT). A insert was then added to the circularized construct to induce expression of the full length antibody, such that the vector would now contain two AOX1 promoters, i.e., one each for heavy and light chain immunoglobulin. The new library was engineered as follows: 0.02 pmol of the linearized construct comprising the pPIC9_IgG1_scFv vector was combined with 0.04 pmol of the promoter insert (SEQ ID NO: 3) in water to a final volume of 5 μL. 5 μL of 2× Gibson Assembly Master Mix (New England Biolabs) was added to the scFv vector / promoter solution. The samples were then incubated at 50° C. for 60 minutes to generate a circularized construct of the promoter insert in the pPIC9_IgG1_...
example 3
Monoclonal Antibody Drug Discovery
[0182]Mice are immunized with a protein or other kind of antigen of interest. Single B cells from the mice are isolated and linked complexes of paired heavy and light chain variable regions are produced in vitro. B cell isolation is performed using droplet microfluidics or isolation into physical containers such as 96-well plates. The fusion construct libraries are inserted into and expressed in host cells on the surface as scFv. The engineered cells are screened for binding to sequence variants of the antigen of interest. RNA or DNA is extracted from engineered cells with binding affinity for the antigen of interest, and the RNA or DNA is sequenced to determine the immunoglobulin content of the selected cells. The linked Ig complexes amplified from the antigen-selected engineered cells are cloned en masse into plasmid vectors to produce a library of plasmid vectors comprising the recombinant fusion construct.
[0183]To study the function of these Ig ...
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