New ligation-independent cloning vectors compatible with a high-throughput platform for parallel construct expression evaluation using baculovirus-infected insect cells.
Biomedical research has undergone a major shift in emphasis over the past decade from characterizing the genomes of organisms to characterizing their proteomes. The high-throughput approaches that were successfully applied to sequencing of genomes, such as miniaturization and automation, have been adapted for high-throughput cloning and protein production. High-throughput platforms allow for a multi-construct, multi-parallel approach to expression optimization and construct evaluation. We describe here a series of baculovirus transfer and expression vectors that contain ligation-independent cloning regions originally designed for use in high-throughput Escherichia coli expression evaluation. These new vectors allow for parallel cloning of the same gene construct into a variety of baculovirus or E. coli expression vectors. A high-throughput platform for construct expression evaluation in baculovirus-infected insect cells was developed to utilize these vectors. Data from baculovirus infection expression trials for multiple constructs of two target protein systems relevant to the study of human diseases are presented. The target proteins exhibit a wide variation in behavior and illustrate the benefit of investigating multiple cell types, fusion partners and secretion signals in optimization of constructs and conditions for eukaryotic protein production.
High-throughput Protein Lab, Center for Structural Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA. wclayb@umich.edu
William Clay
WC
Brown
James
J
DelProposto
J Ronald
JR
Rubin
Kelly
K
Lamiman
Jacob
J
Carless
Janet L
JL
Smith
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D051550:I-kappa B Kinase / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D018411:Spodoptera / Q000378:metabolism* / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D008164:Luminescent Proteins; C100315:NS1 protein, Flavivirus; D017361:Viral Nonstructural Proteins; C054562:yellow fluorescent protein, Bacteria; D051550:I-kappa B Kinase
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