Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture
Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycos...
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| Format: | Conference or Workshop Item |
| Language: | English |
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2005
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| Online Access: | http://eprints.utm.my/3059/3/BPE-41.pdf |
| _version_ | 1825909335963729920 |
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| author | Yap, Wei Ney Abdul Rahman, Badarulhisam Abdul Aziz, Azila |
| author_facet | Yap, Wei Ney Abdul Rahman, Badarulhisam Abdul Aziz, Azila |
| author_sort | Yap, Wei Ney |
| collection | ePrints |
| description | Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyze the galactosylation process by the introduction of �- 1,4galactosyltransferase (�-1,4GalT) as the glycosyltransferase of interest, and uridine-5’- diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of �-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better Nglycan quality. Recombinant �-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine �-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal�14GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and �-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of �-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality. |
| first_indexed | 2024-03-05T18:00:37Z |
| format | Conference or Workshop Item |
| id | utm.eprints-3059 |
| institution | Universiti Teknologi Malaysia - ePrints |
| language | English |
| last_indexed | 2024-03-05T18:00:37Z |
| publishDate | 2005 |
| record_format | dspace |
| spelling | utm.eprints-30592017-08-27T04:57:52Z http://eprints.utm.my/3059/ Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture Yap, Wei Ney Abdul Rahman, Badarulhisam Abdul Aziz, Azila QH301 Biology Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyze the galactosylation process by the introduction of �- 1,4galactosyltransferase (�-1,4GalT) as the glycosyltransferase of interest, and uridine-5’- diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of �-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better Nglycan quality. Recombinant �-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine �-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal�14GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and �-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of �-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality. 2005-12-08 Conference or Workshop Item PeerReviewed application/pdf en http://eprints.utm.my/3059/3/BPE-41.pdf Yap, Wei Ney and Abdul Rahman, Badarulhisam and Abdul Aziz, Azila (2005) Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture. In: 2nd International Conference on Chemical and Bioprocess Engineering in conjunction with 19th Symposium of Malaysian Chemuical Engineers (SOMChE 2005), 8-10 December 2005, Kota Kinabalu, Sabah. |
| spellingShingle | QH301 Biology Yap, Wei Ney Abdul Rahman, Badarulhisam Abdul Aziz, Azila Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| title | Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| title_full | Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| title_fullStr | Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| title_full_unstemmed | Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| title_short | Baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| title_sort | baculovirus coinfection strategy for improved galactosylation of recombinant glycoprotein produced by insect cell culture |
| topic | QH301 Biology |
| url | http://eprints.utm.my/3059/3/BPE-41.pdf |
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