CPC C40B 40/08 (2013.01) [C07K 14/7051 (2013.01); C07K 16/00 (2013.01); C12N 15/1037 (2013.01); C12N 15/1075 (2013.01); C12N 15/1093 (2013.01); C12Q 1/6876 (2013.01); C07K 2317/14 (2013.01); C07K 2317/622 (2013.01); C40B 50/06 (2013.01)] | 10 Claims |
1. A kit for producing a library of constructs for expression of thousands of full-length recombinant immunoglobulins, the kit comprising:
a. a plurality of at least 10,000 unique recombinant fusion polynucleotides, each comprising a first polynucleotide and a second polynucleotide and a linker polynucleotide,
each first polynucleotide encoding a heavy chain variable domain from a cognate pair from a single isolated mammalian B cell and an IgG heavy chain constant domain fragment; and
each second polynucleotide encoding a light chain variable domain from the cognate pair from the single isolated mammalian B cell and an IgK light chain constant domain; and
the linker polynucleotide linking the first and second polynucleotides and comprising a restriction site;
b. a third recombinant polynucleotide, not operationally linked to the recombinant fusion polynucleotides, comprising a first promoter sequence and a sequence encoding a portion of the IgG heavy chain constant domain, wherein the third recombinant polynucleotide comprises a first end sequence and a second end sequence, each overlapping with a first or second end sequence of each recombinant fusion polynucleotide;
c. a fourth recombinant polynucleotide, not operationally linked to the recombinant fusion polynucleotides or the third recombinant polynucleotide, comprising a second promoter sequence or a translational skip site; and
d. a mixture for Gibson assembly.
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