Expression of Sunflower Homeodomain Containing Proteins in Escherichia coli: Purification and Functional Studies

Expression of Sunflower Homeodomain Containing Proteins in Escherichia coli: Purification and... Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competent Escherichia coli cells and induced, the resulting plasmids directed the expression of large amounts (5–10% of total cellular protein) of the encoded polypeptides. As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective absorption onto glutathione–agarose beads, followed by elution with free glutathione. The purified proteins showed both glutathione S -transferase and DNA-binding activity, indicating that they retain their native conformation. The expression–purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells. One of the fusion proteins, RH11 (which is a fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able to bind DNA when spotted onto a nitrocellulose filter. This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate–polyacrylamide gels and used to elicit antibodies which recognized both the transgenic fusion and the native protein from sunflower nuclei. Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Expression and Purification Elsevier

Expression of Sunflower Homeodomain Containing Proteins in Escherichia coli: Purification and Functional Studies

Loading next page...
 
/lp/elsevier/expression-of-sunflower-homeodomain-containing-proteins-in-escherichia-q7RbZjRmit
Publisher
Elsevier
Copyright
Copyright © 1998 Academic Press
ISSN
1046-5928
eISSN
1096-0279
D.O.I.
10.1006/prep.1998.0875
Publisher site
See Article on Publisher Site

Abstract

Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competent Escherichia coli cells and induced, the resulting plasmids directed the expression of large amounts (5–10% of total cellular protein) of the encoded polypeptides. As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective absorption onto glutathione–agarose beads, followed by elution with free glutathione. The purified proteins showed both glutathione S -transferase and DNA-binding activity, indicating that they retain their native conformation. The expression–purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells. One of the fusion proteins, RH11 (which is a fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able to bind DNA when spotted onto a nitrocellulose filter. This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate–polyacrylamide gels and used to elicit antibodies which recognized both the transgenic fusion and the native protein from sunflower nuclei. Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes.

Journal

Protein Expression and PurificationElsevier

Published: Jun 1, 1998

References

  • Homeodomain-DNA recognition
    Gehring, W.J.; Qian, Y.Q.; Billeter, M.; Furukubo-Tokunaga, K.; Schier, A.F.; Resendez-Perez, D.; Affolter, M.; Otting, G.; Wirethrich, K.
  • A cDNA encoding an Hd-Zip protein from sunflower
    Chan, R.L.; Gonzalez, D.H.
  • Isolation and expression pattern of hahr1, from Helianthus annuus.
    Valle, E.M.; Gonzalez, D.H.; Gago, G.M.; Chan, R.L.
  • Developmental and organ-specific changes in promoter DNA–protein interactions in the tomato rbcS
    Manzara, T.; Carrasco, P.; Gruissem, W.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off