Expression of the HSV-1 capsid protein VP19C in Escherichia coli: A single amino acid change overcomes an expression block of the full-length polypeptide

Brandon W. Henson; Nicole Johnson; Alakesh Bera; Mercy E. Okoye; Keshal Viren Desai; Prashant J. Desai

doi:10.1016/j.pep.2010.12.013

Expression of the HSV-1 capsid protein VP19C in Escherichia coli: A single amino acid change overcomes an expression block of the full-length polypeptide

Brandon W. Henson, Nicole Johnson, Alakesh Bera, Mercy E. Okoye, Keshal Viren Desai, Prashant J. Desai

School of Medicine

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.

Original language	English (US)
Pages (from-to)	80-85
Number of pages	6
Journal	Protein Expression and Purification
Volume	77
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2010.12.013
State	Published - May 2011

Keywords

Capsid assembly
Herpes simplex virus
Triplex proteins
VP19C protein expression

ASJC Scopus subject areas

Biotechnology

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title = "Expression of the HSV-1 capsid protein VP19C in Escherichia coli: A single amino acid change overcomes an expression block of the full-length polypeptide",

abstract = "The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.",

keywords = "Capsid assembly, Herpes simplex virus, Triplex proteins, VP19C protein expression",

author = "Henson, {Brandon W.} and Nicole Johnson and Alakesh Bera and Okoye, {Mercy E.} and Desai, {Keshal Viren} and Desai, {Prashant J.}",

note = "Funding Information: Funding for this work was provided by PHS grants from the NIH ( AI R01033077 , AI R01061382 and CA P01113239 ). We thank Augusto Frisancho for help with inserting a Spe1 site in pGEX4T3 and Paul Thompson (Scripps Research Institute, CA) for the modified pET28a plasmid.",

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doi = "10.1016/j.pep.2010.12.013",

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journal = "Protein Expression and Purification",

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T2 - A single amino acid change overcomes an expression block of the full-length polypeptide

AU - Henson, Brandon W.

AU - Johnson, Nicole

AU - Bera, Alakesh

AU - Okoye, Mercy E.

AU - Desai, Keshal Viren

AU - Desai, Prashant J.

N1 - Funding Information: Funding for this work was provided by PHS grants from the NIH ( AI R01033077 , AI R01061382 and CA P01113239 ). We thank Augusto Frisancho for help with inserting a Spe1 site in pGEX4T3 and Paul Thompson (Scripps Research Institute, CA) for the modified pET28a plasmid.

PY - 2011/5

Y1 - 2011/5

N2 - The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.

AB - The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.

KW - Capsid assembly

KW - Herpes simplex virus

KW - Triplex proteins

KW - VP19C protein expression

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Expression of the HSV-1 capsid protein VP19C in Escherichia coli: A single amino acid change overcomes an expression block of the full-length polypeptide

Abstract

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