Vector system for selection of genes encoding secreted proteins and membrane-bound proteins
AssigneeUniversity of South Florida
InventorsJohn P. Cannon, Robert N. Haire, Gary W. Litman
Life of this patent
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Abstract
The subject invention concerns novel vectors for the rapid and robust selection for cDNA sequences that encode secreted or membrane-bound proteins. The invention also pertains to methods for cloning secreted or membrane-bound proteins, including proteins encoded by novel members of gene families.
Claims44
A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises:
- (a) linearizing a vector with one or more restriction enzymes, wherein the vector comprises DNA encoding a reporter molecule lacking a functional signal sequence;
- (b) cutting a candidate nucleic acid sequence with the one or more restriction enzymes and ligating the candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;
- (c) transforming bacterial cells with the ligation product;
- and
- (d) selecting for colonies based on expression of the reporter gene functionally linked to the secreted or membrane-bound protein.
A method for selecting nucleic acid sequences encoding proteins that are secreted or membrane-bound which comprises:
- (a) linearizing a vector with one or more restriction enzymes, wherein the vector comprises DNA encoding β-lactamase lacking a functional signal sequence;
- (b) cutting a candidate nucleic acid sequence with the one or more restriction enzymes and ligating the candidate nucleic acid to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes proteins that are potentially secreted or membrane-bound;
- (c) transforming bacterial cells with the ligation product;
- and
- (d) selecting for colonies based on selection criteria.
A vector for selection of nucleic acid sequences encoding proteins that are secreted or membrane-bound comprising a reporter gene, wherein said reporter gene encodes a reporter molecule lacking a functional signal sequence;
- a selectable marker gene, wherein said reporter gene and said selectable marker gene are operably linked to a promoter sequence;
- and a multiple cloning site.
A vector for selection of nucleic acid sequences encoding proteins that are secreted or membrane-bound comprising a reporter gene, wherein said reporter gene encodes a reporter molecule lacking a functional signal sequence;
- a selectable marker gene, wherein said reporter gene and said selectable marker gene are operably linked to a promoter sequence;
- a nucleotide sequence comprising a plurality of thymidine nucleotides;
- and a multiple cloning site.
A method for selecting nucleic acid sequences encoding secreted or membrane-bound proteins which comprises:
- (a) providing a linearized vector comprising a reporter gene, wherein said reporter gene encodes a reporter molecule lacking a functional signal sequence;
- a selectable marker gene, wherein said reporter gene and said selectable marker gene are operably linked to a promoter sequence;
- and a multiple cloning site;
- (b) ligating a candidate nucleic acid sequence to the linearized vector, thereby forming a ligation product, wherein the candidate nucleic acid sequence encodes a potential secreted or membrane-bound protein;
- (c) transforming bacterial cells with the ligation product;
- and
- (d) selecting for colonies based on expression of the selectable marker gene and the reporter gene functionally linked to the secreted or membrane-bound protein.
Dependencies are inferred from the claim text.