US 12,291,732 B2
Method of producing a sialylated N-glycosylated recombinant protein in periplasm of a recombinant Escherichia coli
Xuejun Hu, Liaoning (CN); Yao Ruan, Liaoning (CN); Ning Ding, Liaoning (CN); Xin Fu, Liaoning (CN); and Jing Zhu, Liaoning (CN)
Assigned to Dalian University, Dalian (CN)
Filed by Dalian University, Liaoning (CN)
Filed on Jun. 3, 2021, as Appl. No. 17/338,525.
Application 17/338,525 is a continuation of application No. PCT/CN2019/072202, filed on Jan. 17, 2019.
Claims priority of application No. 201811615104.8 (CN), filed on Dec. 27, 2018.
Prior Publication US 2021/0292801 A1, Sep. 23, 2021
Int. Cl. C12P 21/00 (2006.01); C12N 9/10 (2006.01); C12N 15/70 (2006.01)
CPC C12P 21/005 (2013.01) [C12N 9/1048 (2013.01); C12N 15/70 (2013.01)] 4 Claims
 
1. A method of producing a sialylated N-glycosylated recombinant protein in the periplasm of a recombinant Escherichia coli, comprising:
(1) constructing an Escherichia coli strain suitable for production of the sialylated N-glycosylated recombinant protein;
(2) constructing an expression system of the sialylated N-glycosylated recombinant protein, comprising:
cloning a glycosyltransferase LsgCDEF gene cluster from Haemophilus influenzae, an undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase WecA gene from Escherichia coli, a oligosaccharide flippase pglK gene from Campylobacter jejuni, an oligosaccharyltransferase pglB gene from Campylobacter jejuni, a sialic-acid synthase NeuBCA gene cluster from Campylobacter jejuni, an α-2,6-sialytransferase Δ16psp2,6ST gene from Vibrionaceae Photobacterium sp. JT-ISH-224, a 332 bp regulatory sequence from upstream of the Pgl gene cluster from Campylobacter jejuni, and a gene of a protein to be modified with a sialylated oligosaccharide chain at the N terminal end, into an Escherichia coli expression vector through genetic recombination; and
(3) transferring the expression system of the sialylated N-glycosylated recombinant protein into the Escherichia coli strain suitable for the production of the sialylated N-glycosylated recombinant protein; and culturing the Escherichia coli strain under auto-induction conditions to produce a protein crude product comprising the sialylated N-glycosylated recombinant protein;
wherein the sialylated oligosaccharide is Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc; and
the 332 bp regulatory sequence is shown in SEQ ID NO: 4.