3 research outputs found
Generation of Recombinant N-Linked Glycoproteins in E. coli
The production of N-linked recombinant glycoproteins is possible in a variety of biotechnology host cells, and more recently in the bacterial workhorse, Escherichia coli. This methods chapter will outline the components and procedures needed to produce N-linked glycoproteins in E. coli, utilizing Campylobacter jejuni glycosylation machinery, although other related genes can be used with minimal tweaks to this methodology. To ensure a successful outcome, various methods will be highlighted that can confirm glycoprotein production to a high degree of confidence, including the gold standard of mass spectrometry analysis
Production of trimeric SARSâCoVâ2 spike protein by CHO cells for serological COVIDâ19 testing
We describe scalable and costâefficient production of full length, Hisâtagged severe acute respiratory syndrome coronavirus 2 (SARSâCoVâ2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARSâCoVâ2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9âfold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GSâCHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9âfold to 53âmg/L. Purification of recombinant spike by Niâchelate affinity chromatography initially yielded a variety of coâeluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzymeâlinked immunosorbent assay format to detect immunoglobulin G antibodies against SARSâCoVâ2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVIDâ19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARSâCoVâ2 trimer which can be used as antigen for mass serological testing
Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome
Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%