Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast both in vitro and in vivo

Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast... A bacterial thermostable cellulase, the endo-1,4-β-D-glucanase E1 from Acidothermus cellulolyticus, was imported into chloroplasts, and an active enzyme was recovered both in vitro and in vivo. Precursor fusion proteins were synthesized with E1 or its catalytic domain, CD, fused to the transit peptide of ferredoxin or ribulose-bisphosphate carboxylase activase for stromal targeting. A spacer region of 1, 5 or 15 amino acids was included carboxy to the transit peptide. The efficiency of import and processing by the stromal processing peptidase depended on the nature of the transit peptide and the passenger protein, and increased with the length of the spacer between them. Besides finding E1 or CD in the stroma, protein was arrested in the envelope during import showing that structural features of E1 and CD, along with their proximity to the transit peptide, influence translocation. The cellulose binding domain and/or serine/proline/threoline-rich linker of E1 may impede efficient import. Significantly, most precursors for E1 and CD synthesized by in vitro translation possessed endoglucanse activity that was temperature-dependent, and required the residues AGGGY at the N-terminus of E1 and CD. Furthermore, activity was detected upon import into chloroplasts. Based on the in vitro analyses, five precursor fusion proteins were selected to determine if E1 and CD would be successfully targeted to chloroplasts in vivo. In transgenic tobacco plants, E1 and CD accumulated in both the stromal and membrane fractions and, importantly, chloroplast extracts showed endoglucanase activity. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast both in vitro and in vivo

Loading next page...
 
/lp/springer_journal/expression-and-import-of-an-active-cellulase-from-a-thermophilic-JBaGykWL0P
Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2003 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1022354124741
Publisher site
See Article on Publisher Site

References

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 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

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

Monthly Plan

  • Read unlimited articles
  • Personalized recommendations
  • No expiration
  • Print 20 pages per month
  • 20% off on PDF purchases
  • Organize your research
  • Get updates on your journals and topic searches

$49/month

Start Free Trial

14-day Free Trial

Best Deal — 39% off

Annual Plan

  • All the features of the Professional Plan, but for 39% off!
  • Billed annually
  • No expiration
  • For the normal price of 10 articles elsewhere, you get one full year of unlimited access to articles.

$588

$360/year

billed annually
Start Free Trial

14-day Free Trial