Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon
View Patent ↗Provided herein is are polypeptides that include the protease domain of a type II transmembrane serine protease (MTSP) as a single chain. Methods using the polypeptides to identify compounds that modulate the protease activity of an MTSP are provided. Also provided are MTSPs designated MTSP3 and MTSP4 and a form of an MTSP designated MTSP6.
1. A nucleic acid molecule, comprising a sequence of nucleic acids encoding a single chain polypeptide that consists only of a protease domain of a type-II membrane-type serine protease (MTSP), or a proteolytically active fragment thereof, as a single chain, wherein:
a free Cys in the protease domain, which is normally disulfide bonded to a Cys in the pro-domain of the full length MTSP, is replaced with another amino acid;
the nucleic acid molecule contains a stop codon so that only the single chain MTSP protease domain, or proteolytically active fragment thereof, can be expressed; and
the MTSP protease domain, or proteolytically active fragment thereof, has serine protease activity as a single chain.
2. The nucleic acid molecule of claim 1 , wherein the MTSP portion has an N-terminus that comprises IVNG, ILGG, VGLL or ILGG.
3. The nucleic acid molecule of claim 1 , wherein the MTSP is selected from among MTSP1, MTSP3, MTSP4 and MTSP6.
4. The nucleic acid molecule of claim 1 , wherein the MTSP protease domain consists of a sequence of amino acid residues selected from among amino acids 615-855 of SEQ ID NO: 2, amino acids 205-437 of SEQ ID NO: 4, the amino acids set forth as SEQ ID NO: 6 or as amino acids 217-443 in SEQ ID NO: 12.
5. The nucleic acid molecule of claim 1 , wherein the encoded polypeptide has at least about 95% sequence identity with a protease domain consisting of a sequence of amino acid residues selected from among amino acids 615-855 of SEQ ID NO: 2, amino acids 205-437 of SEQ ID NO: 4, the amino acids set forth as SEQ ID NO: 6 and amino acids 217-443 in SEQ ID NO: 12.
6. The nucleic acid molecule of claim 1 , wherein the MTSP protease domain is encoded by a sequence of nucleic acid residues selected from among nucleotides 1865-2585 of SEQ ID NO: 1, nucleotides 873-1571 of SEQ ID NO: 3, the nucleotides set forth as SEQ ID NO: 5 and nucleotides 916-1596 of SEQ ID NO: 11.
7. The nucleic acid molecule of claim 1 , wherein a free Cys in the protease domain of the polypeptide is replaced with a serine.
8. The nucleic acid molecule of claim 1 , wherein the MTSP is selected from among corin, MTSP1, enteropeptidase, human airway trypsin-like protease (HAT), TMPRSS2, and TMPRSS4.
9. The nucleic acid molecule of claim 1 , wherein:
the encoded MTSP protease domain is linked directly or via a polypeptide linker to a targeting agent; and
the encoded conjugate has serine protease activity.
10. The nucleic acid molecule of claim 1 , wherein the nucleic acid encoding the MTSP protease domain is operatively linked to a nucleic acid encoding a signal sequence.
11. The nucleic acid molecule of claim 9 , wherein the targeting agent permits:
i) affinity isolation or purification of the conjugate;
ii) attachment of the conjugate to a surface;
iii) detection of the conjugate; or
iv) targeted delivery to a selected tissue or cell.
12. A vector, comprising the nucleic acid molecule of claim 1 .
13. A cell, comprising the vector of claim 12 .
14. The cell of claim 13 that is a eukaryotic cell.
15. The cell of claim 13 that is a yeast cell.
16. The cell of claim 15 that is a Pichia species cell.
17. A method for production of a single chain polypeptide that consists only of a protease domain of a type-II membrane-type serine protease (MTSP), or a proteolytically active fragment thereof, as a single chain, the method comprising:
growing a cell of claim 13 under conditions, whereby the single chain MTSP polypeptide is expressed; and
isolating the single chain MTSP polypeptide.
18. The method of claim 17 , wherein the nucleic acid encoding the MTSP polypeptide is operatively linked to a nucleic acid encoding a signal sequence, whereby the encoded MTSP polypeptide is secreted into the cell culture medium.
19. The method of claim 17 , wherein the cell is a eukaryotic cell.
20. The method of claim 17 , wherein the cell is a yeast cell.
21. The method of claim 20 , wherein the cell is a Pichia species cell.