Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins

Bum Chan Park, Xiang Shen, Michael P Fautsch, Martin Tibudan, Douglas H. Johnson, Beatrice Y J T Yue

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Purpose: To maximize the expression level of myocilin and its truncated proteins in Escherichia coli (E. coli) and to examine the biological effects of bacterially expressed myocilin as compared to eukaryotic myocilin on cultured human trabecular meshwork (TM) cells. Methods: Myocilin full length (1-504 amino acids) and two truncated proteins, myocilin 1-270 and 271-504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin was purified from cultured medium of a transformed TM cell line (TM5) transduced with feline immunodeficiency virus that contains an internal cassette expressing full length myocilin. The morphology and adhesion of human TM cells plated either on fibronectin alone or on fibronectin/ purified myocilin mixtures were assessed by phase contrast microscopy. Actin cytoskeleton was examined using Oregon Green phalloidin. Immunofluorescence staining for paxillin was also performed. Results: The expression of full length and truncated myocilin proteins in Rosetta2(DE3)pLysS was markedly increased especially when the bacteria were grown in media supplemented with 1.0% glucose. Cell adhesion was impaired and microspikes were formed when TM cells were plated onto fibronectin/bacterial full length myocilin mixtures. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was also seen with myocilin 1-270, but not with myocilin 271-504. The eukaryotic full length myocilin produced nearly identical de-adhesive effects as those of the bacterially expressed myocilin. Conclusions: The condition for a high level expression of full length and truncated myocilins in E. coli was optimized. The bacteria] and eukaryotic recombinant full length myocilin produced similar biological consequence on TM cells. The myocilin phenotype appears to be largely due to the NH2-terminal half of the protein.

Original languageEnglish (US)
Pages (from-to)832-840
Number of pages9
JournalMolecular Vision
Volume12
StatePublished - Jul 31 2006

Fingerprint

Proteins
Trabecular Meshwork
Fibronectins
trabecular meshwork-induced glucocorticoid response protein
Paxillin
Feline Immunodeficiency Virus
Escherichia coli
Bacteria
Phenotype
Phalloidine
Phase-Contrast Microscopy
Stress Fibers
Focal Adhesions
Escherichia coli Proteins
Actin Cytoskeleton
Cell Adhesion
Adhesives
Fluorescent Antibody Technique
Actins
Staining and Labeling

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins. / Park, Bum Chan; Shen, Xiang; Fautsch, Michael P; Tibudan, Martin; Johnson, Douglas H.; Yue, Beatrice Y J T.

In: Molecular Vision, Vol. 12, 31.07.2006, p. 832-840.

Research output: Contribution to journalArticle

Park, Bum Chan ; Shen, Xiang ; Fautsch, Michael P ; Tibudan, Martin ; Johnson, Douglas H. ; Yue, Beatrice Y J T. / Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins. In: Molecular Vision. 2006 ; Vol. 12. pp. 832-840.
@article{6d6c64d86a724550b3500fb10382bae2,
title = "Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins",
abstract = "Purpose: To maximize the expression level of myocilin and its truncated proteins in Escherichia coli (E. coli) and to examine the biological effects of bacterially expressed myocilin as compared to eukaryotic myocilin on cultured human trabecular meshwork (TM) cells. Methods: Myocilin full length (1-504 amino acids) and two truncated proteins, myocilin 1-270 and 271-504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin was purified from cultured medium of a transformed TM cell line (TM5) transduced with feline immunodeficiency virus that contains an internal cassette expressing full length myocilin. The morphology and adhesion of human TM cells plated either on fibronectin alone or on fibronectin/ purified myocilin mixtures were assessed by phase contrast microscopy. Actin cytoskeleton was examined using Oregon Green phalloidin. Immunofluorescence staining for paxillin was also performed. Results: The expression of full length and truncated myocilin proteins in Rosetta2(DE3)pLysS was markedly increased especially when the bacteria were grown in media supplemented with 1.0{\%} glucose. Cell adhesion was impaired and microspikes were formed when TM cells were plated onto fibronectin/bacterial full length myocilin mixtures. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was also seen with myocilin 1-270, but not with myocilin 271-504. The eukaryotic full length myocilin produced nearly identical de-adhesive effects as those of the bacterially expressed myocilin. Conclusions: The condition for a high level expression of full length and truncated myocilins in E. coli was optimized. The bacteria] and eukaryotic recombinant full length myocilin produced similar biological consequence on TM cells. The myocilin phenotype appears to be largely due to the NH2-terminal half of the protein.",
author = "Park, {Bum Chan} and Xiang Shen and Fautsch, {Michael P} and Martin Tibudan and Johnson, {Douglas H.} and Yue, {Beatrice Y J T}",
year = "2006",
month = "7",
day = "31",
language = "English (US)",
volume = "12",
pages = "832--840",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins

AU - Park, Bum Chan

AU - Shen, Xiang

AU - Fautsch, Michael P

AU - Tibudan, Martin

AU - Johnson, Douglas H.

AU - Yue, Beatrice Y J T

PY - 2006/7/31

Y1 - 2006/7/31

N2 - Purpose: To maximize the expression level of myocilin and its truncated proteins in Escherichia coli (E. coli) and to examine the biological effects of bacterially expressed myocilin as compared to eukaryotic myocilin on cultured human trabecular meshwork (TM) cells. Methods: Myocilin full length (1-504 amino acids) and two truncated proteins, myocilin 1-270 and 271-504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin was purified from cultured medium of a transformed TM cell line (TM5) transduced with feline immunodeficiency virus that contains an internal cassette expressing full length myocilin. The morphology and adhesion of human TM cells plated either on fibronectin alone or on fibronectin/ purified myocilin mixtures were assessed by phase contrast microscopy. Actin cytoskeleton was examined using Oregon Green phalloidin. Immunofluorescence staining for paxillin was also performed. Results: The expression of full length and truncated myocilin proteins in Rosetta2(DE3)pLysS was markedly increased especially when the bacteria were grown in media supplemented with 1.0% glucose. Cell adhesion was impaired and microspikes were formed when TM cells were plated onto fibronectin/bacterial full length myocilin mixtures. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was also seen with myocilin 1-270, but not with myocilin 271-504. The eukaryotic full length myocilin produced nearly identical de-adhesive effects as those of the bacterially expressed myocilin. Conclusions: The condition for a high level expression of full length and truncated myocilins in E. coli was optimized. The bacteria] and eukaryotic recombinant full length myocilin produced similar biological consequence on TM cells. The myocilin phenotype appears to be largely due to the NH2-terminal half of the protein.

AB - Purpose: To maximize the expression level of myocilin and its truncated proteins in Escherichia coli (E. coli) and to examine the biological effects of bacterially expressed myocilin as compared to eukaryotic myocilin on cultured human trabecular meshwork (TM) cells. Methods: Myocilin full length (1-504 amino acids) and two truncated proteins, myocilin 1-270 and 271-504, were expressed and purified from an E. coli strain, Rosetta2(DE3)pLysS. The eukaryotic myocilin was purified from cultured medium of a transformed TM cell line (TM5) transduced with feline immunodeficiency virus that contains an internal cassette expressing full length myocilin. The morphology and adhesion of human TM cells plated either on fibronectin alone or on fibronectin/ purified myocilin mixtures were assessed by phase contrast microscopy. Actin cytoskeleton was examined using Oregon Green phalloidin. Immunofluorescence staining for paxillin was also performed. Results: The expression of full length and truncated myocilin proteins in Rosetta2(DE3)pLysS was markedly increased especially when the bacteria were grown in media supplemented with 1.0% glucose. Cell adhesion was impaired and microspikes were formed when TM cells were plated onto fibronectin/bacterial full length myocilin mixtures. Loss of actin stress fibers and focal adhesions was also observed. This myocilin phenotype was also seen with myocilin 1-270, but not with myocilin 271-504. The eukaryotic full length myocilin produced nearly identical de-adhesive effects as those of the bacterially expressed myocilin. Conclusions: The condition for a high level expression of full length and truncated myocilins in E. coli was optimized. The bacteria] and eukaryotic recombinant full length myocilin produced similar biological consequence on TM cells. The myocilin phenotype appears to be largely due to the NH2-terminal half of the protein.

UR - http://www.scopus.com/inward/record.url?scp=33746755259&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33746755259&partnerID=8YFLogxK

M3 - Article

VL - 12

SP - 832

EP - 840

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -