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Vector system for selection of genes encoding secreted proteins and membrane-bound proteins

a gene selection and membrane-bound protein technology, applied in the field of vector system for selecting genes encoding secreted proteins and membrane-bound proteins, can solve the problems of low transfection efficiency, signal sequence trapping using mammalian cells, and difficult identification of secretion signal sequences that are normally present in the 5′ end of cdna encoding, so as to improve the overall efficiency of cloning cdnas

Inactive Publication Date: 2007-05-17
UNIV OF SOUTH FLORIDA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a vector system that allows for the rapid and robust selection of cDNAs that encode secreted or membrane-bound proteins. The vector system includes a reporter gene lacking a functional signal sequence, a selectable marker gene, and a multiple cloning site. The vector system can also include a SLIP sequence and thymidine nucleotides to increase the efficiency of cloning cDNAs for secreted or membrane-bound proteins. The invention also includes a method for cloning novel members of a gene family using the vector system. The vector system has been validated with known members of the Ig gene superfamily and has led to the identification of a novel gene in amphioxus.

Problems solved by technology

However, ESTs are small random cDNA sequences and thus it becomes hard to identify secretion signal sequence that is normally present in the 5′ end of cDNA encoding secreted protein.
Signal sequence trapping using mammalian cells has disadvantages, including low transfection efficiency, relatively expensive culture medium, and difficult recovery of vector-borne cDNA sequences from cells that have been transfected.
Signal sequence trapping using yeast cells also has the disadvantage of slow growth time as compared to bacterial cells.
Further, methods for molecular cloning in yeast cells are generally more complicated than bacterial methods.
It does not teach identifying signal sequences in complex eukaryotic organisms.
Further, none of the aforementioned systems incorporate a single, degenerate primer-based polymerase chain reaction (PCR) strategy designed to clone novel gene family members.

Method used

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  • Vector system for selection of genes encoding secreted proteins and membrane-bound proteins
  • Vector system for selection of genes encoding secreted proteins and membrane-bound proteins
  • Vector system for selection of genes encoding secreted proteins and membrane-bound proteins

Examples

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Effect test

example 1

Amplification of cDNAs Encoding Secreted or Membrane-Bound Proteins Containing Specific Amino Acid Motifs

[0066] In order to select cDNAs that encode secreted or membrane-bound proteins, the cloning vector G7311 (referred to herein as an “Amptrap” vector) was designed by the present inventors, which allows selection for signal sequence-encoding regions by β-lactamase rescue (as shown in FIG. 2). The Amptrap vector shown contains a 5′-truncated β-lactamase gene driven by a Lac promoter. The product of the modified β-lactamase gene lacks the signal peptide present in the wild-type protein and is therefore unable to be exported into the bacterial periplasmic space. An asymmetric pair of SfiI sites for insertion of cDNA lies immediately 5′ to the β-lactamase open reading frame (ORF). A separate neomycin phosphotransferase marker was included to allow propagation of the vector without cDNA inserts. Because β-lactamase must be secreted into the periplasmic space to produce ampicillin resi...

example 2

Cloning of Expressed Sequence Tags (EST) Sequences Using the Amptrap Vector

[0068] cDNA was synthesized from Florida lancelet (Branchiostoma floridae) and sea lamprey (Petromyzon marinus) tissues and cloned into an Amptrap vector. 57 sequences were analyzed by BLASTX searching of the Genbank database. Although 17 sequences failed to match known sequences in Genbank, all of the remaining 40 sequences were found to encode proteins that are likely to be secreted or bound to membranes (Table 1).

TABLE 1Beta-lactamase fusion (“amptrap”) EST sequencesG#TissueSourceStock?Gel dateComments7024PMP20000827, clone 3Y20000928no match*7026PMI20000827, clone 5Y20000830trypsinogen b1†7027PMI20000827, clone 6Y20000830no match*7031PMI20000827, clone 10Y20000830trypsinogen b1†7033PMI20000827, clone 12Y20000830trypsinogen b1†7034PMI20000827, clone 13Y20000905trypsinogen b1†7035PMI20000827, clone 14Y20000905chymotrypsinogen†7037PMI20000827, clone 16Y20000905trypsinogen†7038PMI20000827, clone 17Y2000090...

example 3

Amplification of Candidate Immune-Type Receptor Genes from Branchiostoma floridae, Raja eglanteria, and Petromyzon marinus

[0069] In order to identify potential new members of the novel immune-type receptor (NITR) gene family previously described in teleost fish, cDNA sequences from Florida lancelet Branchiostoma floridae, clearnose skate Raja eglanteria, and sea lamprey Petromyzon marinus tissues were amplified by 5′-RACE PCR using various 3′ primers and the 5′-SMART oligonucleotide. These primers included:

(SEQ ID NO. 8)nitrVYWFR-5′TGGCCGAGGCGGCCCNCGRAACCARTANAC-3′;Sfi:(SEQ ID NO. 9)nitrVYWF-Sfi:5′GACTGGCCGAGGCGGCCCRAACCARTANAC-3′;(SEQ ID NO. 10)nitrYWFR-Sfi:5′-GACTGGCCGAGGCGGCCCNCGRAACCARTA-3′;(SEQ ID NO. 11)nitrYWFK-Sfi:5′-GACTGGCCGAGGCGGCCCYTTRAACCARTA-3′;(SEQ ID NO. 12)nitrWFR1-Sfi:5′-GACTGGCCGAGGCGGCCCNCGRAACCA-3′;(SEQ ID NO. 13)nitrWFR2-Sfi:5′-GACTGGCCGAGGCGGCCCYCTRAACCA-3′;and(SEQ ID NO. 14)nitrWFK-Sfi:5′GACTGGCCGAGGCGGCCCYTTRAACCA-3′.

[0070] The pool of amplicons were subs...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation of U.S. application Ser. No. 10 / 138,998, filed May 2, 2002, which claims the benefit of U.S. Provisional Application Ser. No. 60 / 288,046, filed May 2, 2001, each of which is hereby incorporated by reference in its entirety, including all nucleic acid sequences, amino acid sequences, figures, tables, and drawings.[0002] The subject invention was made with government support under a research project supported by National Institutes of Health Grant No. AI23338. The government may have certain rights in this invention.BACKGROUND OF THE INVENTION [0003] Proteins destined for transport into or across cell membranes are usually translated with a signal sequence that directs the newly synthesized protein to the appropriate membrane translocation system. The primary structure of signal sequences is highly variable among different proteins. Signal sequences that target proteins for export from the cytosol...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/06C12N9/16C12N9/80C12N1/21C07H21/04C12N15/74C12N15/10C12P19/34C12Q1/68
CPCC12N15/1051
Inventor CANNON, JOHN P.HAIRE, ROBERT N.LITMAN, GARY W.
Owner UNIV OF SOUTH FLORIDA
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