US 12,291,733 B2
Process for the preparation of recombinant lectin protein
Dhananjay Sathe, Maharashtra (IN); Sudeep Kumar, Gujarat (IN); and Mamata Katdare, Maharashtra (IN)
Assigned to Unichem Laboratories Ltd, Maharashtra (IN)
Appl. No. 17/273,044
Filed by Unichem Laboratories Ltd, Maharashtra (IN)
PCT Filed Sep. 5, 2019, PCT No. PCT/IB2019/057471
§ 371(c)(1), (2) Date Mar. 3, 2021,
PCT Pub. No. WO2020/074977, PCT Pub. Date Apr. 16, 2020.
Claims priority of application No. 201821008382 (IN), filed on Sep. 7, 2018; and application No. 201921001559 (IN), filed on Jan. 14, 2019.
Prior Publication US 2021/0324438 A1, Oct. 21, 2021
Int. Cl. C12P 21/02 (2006.01); B01D 15/32 (2006.01); B01D 15/36 (2006.01); C07K 1/18 (2006.01); C07K 1/20 (2006.01); C07K 1/34 (2006.01); C07K 1/36 (2006.01); C07K 14/42 (2006.01); C07K 14/47 (2006.01)
CPC C12P 21/02 (2013.01) [B01D 15/327 (2013.01); B01D 15/362 (2013.01); B01D 15/363 (2013.01); C07K 1/18 (2013.01); C07K 1/20 (2013.01); C07K 1/34 (2013.01); C07K 1/36 (2013.01); C07K 14/42 (2013.01); C07K 14/4726 (2013.01)] 13 Claims
 
1. A method of preparing a recombinant lectin protein with improved cleavage of the initiator methionine, the method comprising expressing a recombinant lectin protein encoded by a recombinant lectin gene in a host cell in culture, wherein
a. an induction phase is initiated by adding an inducer to the culture;
b. the host cell is a recombinant E. coli cell, and expression during the induction phase is performed under conditions that cause the host cell to have a doubling time between 100 minutes and 160 minutes, wherein said conditions include the following:
c. the induction phase is carried out at a temperature of at least 15° C. and no more than 22° C., and carbon to nitrogen ratio is maintained during the expression phase from 3:1 to 6:1; wherein the recombinant lectin protein is selected from
i. SEQ ID NO:1;
ii. SEQ ID NO:3;
iii. SEQ ID NO:4; or
iv. an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% homology with i, ii, or iii;
d. the induction phase is carried out for at least 10 hours and the optical density of the culture is at least 25 and no more than 40;
e. carbon source is added to the culture at a rate of from 0.5 to 2 gL-1h-1;
f. the nitrogen source is added to the culture at a rate of from 0.4 to 1.5 gL” 1h-1;
g. an inducer concentration of at least 0.1 mM and no more than 0.5 mM wherein the inducer is IPTG (isopropyl thio-galacto-pyranoside);
h. purifying the crude recombinant lectin protein using at least one chromatography step,
wherein by carrying out the induction phase under conditions e.g., resulting in a doubling time of 100 to 160 hours, cleavage of the initiator methionine is catalysed to reduce methionine residue in the recombinant lectin protein, and obtain a recombinant lectin protein in which less than 20% of recombinant lectin includes the initiator methionine.