Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

Anders Hafren, Kristiina Mäkinen

Research output: Contribution to journalArticleScientificpeer-review

Abstract

In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3' end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and Nla forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pl 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. Nlb, Cl and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.
Original languageEnglish
JournalJournal of General Virology
Volume89
Issue number6
Pages (from-to)1509-1518
Number of pages10
ISSN0022-1317
DOIs
Publication statusPublished - 2008
MoE publication typeA1 Journal article-refereed

Cite this

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title = "Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein",
abstract = "In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3' end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and Nla forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pl 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. Nlb, Cl and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.",
author = "Anders Hafren and Kristiina M{\"a}kinen",
year = "2008",
doi = "10.1099/vir.0.83649-0",
language = "English",
volume = "89",
pages = "1509--1518",
journal = "Journal of General Virology",
issn = "0022-1317",
publisher = "American Society for Microbiology",
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}

Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein. / Hafren, Anders; Mäkinen, Kristiina.

In: Journal of General Virology, Vol. 89, No. 6, 2008, p. 1509-1518.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

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AU - Mäkinen, Kristiina

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N2 - In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3' end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and Nla forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pl 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. Nlb, Cl and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.

AB - In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3' end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and Nla forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pl 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. Nlb, Cl and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.

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