Cloning, sequencing, and biochemical characterization of endo-N-acylneuraminidase

Bacteriophage K1F Endo-N-acylneuraminidase hydrolyzes alpha-2,8-linked polysialic acid found on K1 encapsulated Escherichia coli and on the neural cell adhesion molecule (NCAM). A novel cloning strategy, which pursued ligation of partially restricted ALU 1 or SAU 3A genomic K1F DNA fragments, is described. Sequence analysis reveals that Endo-N is a domainally arranged, 920 amino acid protein with a T7-like gene organization. A 200 amino acid N-terminal domain is homologous to the T7 tail protein N-terminus (gene 17). A 520 amino acid middle domain is possibly similar to the Sea Urchin arylsulfatase precursor, as determined by statistical processing of jumbled sequences. Results from comparative analyses of epitope and nucleotide similarities to PK1E endoneuraminidase indicate that a 200 amino acid C-terminal domain, unique to K1F Endo-N, could be responsible for stabilizing K1F Endo-N to proteolysis. In addition to sequence analysis, biochemical characterization indicates that the molecule is a dimer of identical 102 kDa polypeptide chains. Immunoblot analysis of SDS-PAGE fractionated Endo-N indicates that no post-transcriptional modifications are necessary for expression of native protein. A method for detecting molecular weight variants, which could be indicative of in vivo protein folding intermediates as found in the P22 tail spike protein, is presented. Future research with Endo-N depends on developing an expression vector. Biochemical and nucleotide sequence results presented herein indicate that construction of an Endo-N expression vector is possible.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Veterinary Medical Science

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Petter, Jean Guard

Contributors dc:contributor

• Vimr, Eric R.

• Biology, Molecular
• Biology, Neuroscience
• Chemistry, Biochemistry

Statement dc:rights

• Copyright 1991 Petter, Jean Guard

Language dc:language

eng

Identifier

AAI9136702
(UMI)AAI9136702

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/19202