Development of a Repository for Glycan-related Enzymes

Project: Research project

Description

Design and preparation of expression constructs: Three parallel recombinant expression platforms will be pursued to provide the greatest opportunity for functional protein production in one of the three host systems. The Gateway recombination platform allows the capture of a given coding region or domain-encoding cassette as an entry vector and the subsequent transfer into a variety of destination vectors for expression in the respective recombinant host platforms. Full-length cDNA clones encoding ~90% of the human GT and GH sequences were identified within the MGC repository and sequence alignments within enzyme families have been initiated for the design of appropriate truncated expression constructs. Catalytic domain sequences will be amplified and captured as entry vector constructs using a modification of the strategy developed in the LaBaer lab for the generation of full-length expression-ready gene collections (FLEXGene) of human kinases and breast cancer-related genes[13, 15], the genomes of S. cerevisiae[10], Y. pestis[12], F. tularensis[12], P. aeruginosa[11], and V. cholerae[14]. Coding regions will be amplified using primer sets that incorporate a tobacco etch virus (TEV) protease recognition site into either the NH2- or COOH-terminus of the respective GT or GH sequence and att recombination sites will also be engineered to flank the amplimer product. Amplification products will be captured using the standard Gateway BP reaction for coding regions 2000bp will be captured using the In-Fusion protocol. In both strategies the amplification products will be generated in the Gateway pDONR221 entry vector as fusions to a TEV protease recognition site and verified by full-length sequence analysis. Gateway destination vectors will be designed and synthesized for expression in mammalian cells (pEAK10 episomal mammalian expression vector, Edge Biosystems), baculovirus/insect cells (BaculoDirect baculovirus vector system, Invitrogen), and E. coli (pET E. coli expression vectors, Novagen). For each expression platform a pair of destination vectors will be prepared to accept inserts either as NH2- terminal or COOH-terminal fusions depending on the nature of the entry clone sequence.enu_13
StatusFinished
Effective start/end date9/24/099/23/12

Funding

  • HHS-NIH-NCATS: National Center for Research Resources (NCRR): $270,863.00

Fingerprint

Baculoviridae
Genetic Recombination
Polysaccharides
Clone Cells
Escherichia coli
Sequence Alignment
Cholera
Neoplasm Genes
Enzymes
Sequence Analysis
Saccharomyces cerevisiae
Insects
Catalytic Domain
Phosphotransferases
Complementary DNA
Genome
Breast Neoplasms
Gene Expression
Proteins
TEV protease