A desiccation-tolerant cyanobacterium <i>Nostoc commune</i> accumulates a novel group of water stress proteins (Wsp) in response to cycles of repeated drying and rehydration. Antibodies, specific for Wsp, were used to screen a lambdafix II library of <i>N. commune</i> UTEX 584 Bam H1 DNA fragments and an 8.5-kb fragment, containing a gene cluster that synthesized a 59-kDa cross-reactive protein. The cloned fragment comprised five ORF’s. The ORF’s 59, 24, 22, 36, and 70, each potentially encode products of molecular weights of 59, 24, 22, 36, and 70-kDa, respectively. The 59 and 24 ORF products were found to be expressed in <i>E. coli</i>. The 59-kDa product of this fragment gives the strongest cross-reaction with the Wsp antiserum. The 59-kDa protein was partially purified. The 24-kDa product was successfully purified to homogeneity and partially characterized.
This study used <i>E. coli</i> strain DH10B transformed with the pTrc 99A plasmid. The pTre 99A contains the 8.5-kb gene cluster fragment of interest. The products of ORF 24 and 59 were isolated using an initial 40-60 % ammonium sulfate precipitation of a clarified <i>E. coli</i> cell lysate. The clarified cell lysate was then subjected to streptomycin sulfate precipitation. The cell lysate was then dialyzed extensively. The cell lysate was then applied to a Mono Q HR 5/5 anion exchange column using a 2 M KCl gradient elution procedure. The Mono Q column yielded a fraction containing both ORF products which eluted with approximately 400 mM KCl. This fraction was then applied to a Superose 12 HR 10/30 gel filtration column. The eluent fraction containing the ORF 24 product was then reapplied to the Superose 12 to yield the final fraction containing only the ORF 24 product. The final fraction of ORF 24 was purified to homogeneity as determined by SDS-PAGE analysis. Approximately 750 μg of ORF 24 was isolated. This preparation was used for characterization studies.
Characterization studies of ORF 24 consisted of an amino-terminal sequence analysis, an estimation of the molecular weight using gel filtration chromatography and SDS-PAGE analysis, and an analysis of enzymatic activity as suggested by amino acid sequence homologies. The amino-terminal sequence of ORF 24 is P V E Q R S H D. The molecular weight of ORF 24 using gel-filtration chromatography and SDS-PAGE analysis is 26-kDa and 23-kDa, respectively. From gene sequence analysis, the molecular weight of ORF 24 is known to be 24,340-Da. These data indicate that ORF 24 is a monomer. ORF 24 was found to have amino acid sequence homologies with a pectate lyase (E 4.2.2.2) periplasmic precursor from <i>Erwinia caratovora</i> subspecies and a dextransucrase (EC 2.4.1.5) precursor from <i>Streptoccocus mutans</i> GS-5. However, pectate lyase activity was not detected in cellular extracts over a 24 hour period. In addition, ORF 24 was not found to interact with 10 % substrate solutions of N-acetylglucosamine, pectin, UTEX 584 sheath material, DRH1 sheath material, sucrose, or glucose using thin layer chromatography. These studies indicate that the enzymatic activities proposed from amino acid sequence homologies have not been detected. The suggestion that ORF 24 is a water stress protein with a protective function on a structural level with regards to desiccation-tolerance requires further study. / Master of Science
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/46434 |
| Date | 30 December 2008 |
| Creators | Sines, Brian James |
| Contributors | Chemical Engineering, Bevan, D.R., Potts, Malcolm, Velander, William H., Conger, William L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | ix, 48 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 35217366, LD5655.V855_1996.S546.pdf |
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