	US 12,234,266 B2
	CRM197 protein expression
I-Ming Cho, Taipei (TW); Chia-Hung Chiu, Taipei (TW); and Lee-Cheng Liu, Taipei (TW)
Assigned to OBI PHARMA, INC., Taipei (TW)
Appl. No. 16/962,992
Filed by OBI PHARMA, INC., Taipei (TW)
PCT Filed Jan. 18, 2019, PCT No. PCT/US2019/014156 § 371(c)(1), (2) Date Jul. 17, 2020, PCT Pub. No. WO2019/143911, PCT Pub. Date Jul. 25, 2019.
Claims priority of provisional application 62/619,449, filed on Jan. 19, 2018.
Prior Publication US 2023/0018689 A1, Jan. 19, 2023
Int. Cl. C07K 14/34 (2006.01); C12N 15/70 (2006.01)

CPC C07K 14/34 (2013.01) [C12N 15/70 (2013.01); C07K 2319/02 (2013.01); C07K 2319/034 (2013.01)]

5 Claims

1. A method of producing a CRM197 protein, comprising culturing a cell comprising an expression plasmid having a polynucleotide, wherein the polynucleotide comprises:

(a) a CRM197 nucleotide sequence comprising SEQ ID NO: 1, and

(b) a secretion signal sequence encoding amino acids of SEQ ID NO: 2 at 5′-terminus of the CRM197 nucleotide sequence; and

inducing expression of the CRM197 protein, wherein the induction is performed at pH 7.5-8.0 in an expression medium, and an expression temperature of the CRM197 protein is first incubating at 37° C. and then reducing to about 20-25° C.,

wherein the expression plasmid comprises a phosphate regulated promoter, and the cell comprises E. coli,

wherein the phosphate regulated promoter is a bacterial alkaline phosphatase A promoter; and the CRM197 protein is soluble, periplasmic, and folded and comprises two intra-disulfide bonds.