US 12,351,628 B2
Modified membrane spanning proteins and methods for the preparation and use thereof
Mauro Mileni, San Diego, CA (US)
Assigned to ABILITA THERAPEUTICS, INC., San Diego, CA (US)
Filed by Abilita Bio, Inc., San Diego, CA (US)
Filed on Jun. 1, 2021, as Appl. No. 17/335,677.
Application 17/335,677 is a continuation of application No. 16/411,059, filed on May 13, 2019, granted, now 11,053,312.
Application 16/411,059 is a continuation of application No. 14/928,128, filed on Oct. 30, 2015, granted, now 10,287,349, issued on May 14, 2019.
Claims priority of provisional application 62/073,554, filed on Oct. 31, 2014.
Prior Publication US 2021/0363239 A1, Nov. 25, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 16/28 (2006.01); C07K 14/705 (2006.01); C12N 15/10 (2006.01); A61K 38/00 (2006.01)
CPC C07K 16/28 (2013.01) [C07K 14/705 (2013.01); C12N 15/1037 (2013.01); C12N 15/1079 (2013.01); C12N 15/1093 (2013.01); A61K 38/00 (2013.01)] 17 Claims
 
1. A method of modulating one or more functional properties of a membrane-spanning protein, said method comprising:
(a) generating a first polynucleotide library from a starting polynucleotide that encodes the membrane-spanning protein, wherein a sufficient number of bases of said starting polynucleotide are randomly modified so as to modulate the one or more functional properties of the membrane-spanning protein,
(b) generating a second polynucleotide library from said first polynucleotide library by DNA shuffling, said second polynucleotide library comprising a modified polynucleotide encoding a modified membrane-spanning protein comprising said one or more modulated functional properties,
(c) inserting the modified polynucleotides of said second polynucleotide library into a construct comprising:
a signal sequence,
a first marker sequence, wherein said first marker sequence is in-frame with said modified polynucleotide, is downstream of said modified polynucleotide, and encodes a polypeptide that overcomes the sensitivity of a host cell to the presence of a first selective pressure agent or condition and the polypeptide is fused to said modified membrane-spanning protein, and
a second marker sequence, wherein said second marker sequence is in-frame and downstream of said signal sequence, but upstream of said modified polynucleotide, is different from the first marker sequence and encodes a polypeptide that overcomes the sensitivity of a host cell to the presence of a second selective pressure agent or condition, and is fused to said modified membrane-spanning protein, thereby producing modified polynucleotide-containing constructs,
(d) transforming a plurality of host cells with the modified polynucleotide-containing constructs to obtain a plurality of transformed host cells;
(e) culturing the transformed host cells in the presence of said first and second selective pressure agents/conditions, wherein the concentration/condition levels of said first and second selective pressure agents/conditions are higher than the concentration/condition levels of said first and second selective pressure agents/conditions used for culturing an equivalent host cell transformed with the starting polynucleotide;
(f) selecting a host cell from step (e) that survives exposure to said first and second selective pressure agents/conditions; and
(g) identifying the modified polynucleotide-containing construct(s) contained therein;
wherein the surviving host cells from step (f) express the modified membrane-spanning protein having the one or more modulated functional properties relative to the membrane-spanning protein encoded by the starting polynucleotide.