Expressed sequence tags and encoded human proteins
View Patent ↗The sequences of 5′ ESTs derived from mRNAs encoding secreted proteins are disclosed. The 5′ ESTs may be to obtain cDNAs and genomic DNAs corresponding to the 5′ ESTs. The 5′ ESTs may also be used in diagnostic, forensic, gene therapy, and chromosome mapping procedures. Upstream regulatory sequences may also be obtained using the 5′ ESTs. The 5′ ESTs may also be used to design expression vectors and secretion vectors.
1. A purified and isolated nucleic acid sequence encoding a polypeptide comprising amino acids −26 to −1 of SEQ ID NO: 2259 fused in frame to the 5′ end of a polynucleotide sequence encoding a heterologous polypeptide.
2. The purified and isolated nucleic acid sequence of claim 1 , wherein said nucleic acid sequence comprises nucleotides 170 to 247 of SEQ ID NO: 540.
3. A method of making a cDNA encoding a human secreted protein wherein said method comprises die steps of:
a) contacting a collection of mRNA molecules from human cells with a primer comprising at least 15 consecutive nucleotides of a sequence complementary to a sequence of SEQ ID NO: 540;
b) hybridizing said primer to an mRNA in said collection;
c) reverse transcribing said hybridized primer to make a first cDNA strand from said mRNA;
d) making a second cDNA strand complementary to said first cDNA strand; and
e) isolating the resulting cDNA encoding said polypeptide comprising said first cDNA strand and said second cDNA strand.
4. A method of making a cDNA encoding a human secreted protein comprising the steps of:
a) obtaining a cDNA comprising a sequence of SEQ ID NO: 540;
b) contacting said cDNA with a detectable probe comprising at least 15 consecutive nucleotides of said sequence of SEQ ID NO: 540 or a sequence complementary thereto under conditions which permit said probe to hybridize to said cDNA;
c) identifying a cDNA which hybridizes to said detectable probe; and
d) isolating said cDNA which hybridizes to said probe.
5. A method of making a cDNA encoding a human secreted protein comprising the steps of:
a) contacting a collection of mRNA molecules from human cells with a first primer capable of hybridizing to the polyA tail of said mRNA;
b) hybridizing said first primer to said polyA tail;
c) reverse transcribing said mRNA to make a first cDNA strand;
d) making a second cDNA strand complementary to said first cDNA strand using at least one punier comprising at least 15 nucleotides of a sequence SEQ ID NO:540; and
e) isolating the resulting cDNA comprising said first cDNA strand and said second cDNA strand.
6. A method of making a polypeptide comprising the steps of:
a) introducing into a host cell or organism a nucleic acid sequence encoding a polypeptide comprising amino acids −26 to −1 of SEQ ID NO: 2259 fused in frame to the 5′ end of a polynucleotide sequence encoding a heterologous polypeptide
b) providing conditions that allow for the expression and secretion of said polypeptide by said host cell, organism, or tissue.
7. The method of claim 6 , wherein said nucleic acid is operably linked to a promoter.
8. The method of claim 6 , further comprising the recovery of said expressed and secreted polypeptide.
9. The method of claim 8 , wherein the step of isolating the expressed and secreted protein comprises purifying the secreted protein from the supernatant, the culture medium or the cell extract of said host cell, organism, or tissue.
10. The method of claim 6 , wherein said nucleic acid encodes a polypeptide comprising amino acids −26 to −1 of SEQ ID NO: 2259.
11. A vector comprising a nucleic acid encoding a polypeptide comprising amino acids −26 to −1 of SEQ ID NO: 2259 fused in frame to the 5′ end of a polynucleotide sequence encoding a heterologous polypeptide.
12. A purified and isolated nucleic acid sequence encoding a polypeptide comprising:
a) amino acids −1 to 29 of SEQ ID NO: 2259; or
b) amino acids −26 to 29 of SEQ ID NO: 2259.
13. The purified and isolated nucleic acid sequence of claim 12 , wherein said nucleic acid sequence encodes amino acids 1 to 29 of SEQ ID NO: 2259.
14. The purified and isolated nucleic acid sequence of claim 12 , wherein said nucleic acid sequence encodes amino acids −26 to 29 of SEQ ID NO: 2259.
15. The purified and isolated nucleic acid sequence of claim 14 , wherein said nucleic acid sequence comprises nucleotides 170 to 334 of SEQ ID NO: 540.
16. A vector comprising a nucleic acid sequence encoding a polypeptide comprising:
a) amino acids 1 to 29 of SEQ ID NO: 2259; or
b) amino acids −26 to 29 of SEQ ID NO: 2259.
17. The vector of claim 16 , wherein said nucleic acid sequence encodes amino acids 1 to 29 of SEQ ID NO: 2259.
18. The vector of claim 16 , wherein said nucleic acid sequence encodes amino acids −26 to 29 of SEQ ID NO: 2259.
19. The vector of claim 16 , wherein said nucleic acid sequence comprises nucleotides 170 to 334 of SEQ ID NO: 540.
20. A method of making a polypeptide comprising:
a) introducing into a host cell or organism a nucleic acid sequence encoding a polypeptide comprising:
i)amino acids 1 to 29 of SEQ ID NO: 2259; or
ii)amino acids −26 to 29 of SEQ ID NO: 2259; and
b) providing conditions tat allow for the expression and secretion of said polypeptide by said host cell, organism, or tissue.
21. The method of claim 20 , wherein said nucleic acid is operably linked to a promoter.
22. The method of claim 20 , wherein said nucleic acid encodes amino acids 1 to 29 of SEQ ID NO: 2259.
23. The method of claim 22 , wherein said nucleic acid sequence is operably linked to a promoter.
24. The method of claim 20 , wherein said nucleic acid encodes amino acids −26 to 29 of SEQ ED NO: 2259.
25. The method of claim 24 , wherein said nucleic acid sequence is operably linked to a promoter.
26. The method of claim 25 , wherein the step of isolating the expressed and secreted protein comprises purifying the secreted protein from the supernatant, the culture medium or the cell extract of said host cell, organism, or tissue.
27. The method of claim 20 , further comprising the recovery of said expressed and secreted polypeptide.