Methods, compositions and cells for preparing surfactant protein D (SP-D)
Some embodiments of the methods and compositions provided herein relate to the preparation surfactant protein-D (SP-D). Some embodiments include the expression of human SP-D in certain cell lines, and the purification of human SP-D from such cell lines. Some embodiments include the preparation of certain oligomeric forms of human SP-D.
1. A method for producing a human surfactant protein D (SP-D) polypeptide composition comprising:
(a) introducing a polynucleotide encoding the SP-D polypeptide into a human cell selected from the group consisting of: NM-H9D8, NM-H9D8-E6Q12, NM-H9D8(8B11), and NM-F9;
(b) culturing the cell under conditions in which the SP-D polypeptide is expressed; and
(c) isolating the expressed SP-D polypeptide from the cell, wherein the expressed SP-D polypeptide comprises a glycosylation pattern substantially similar to a glycosylation pattern of a naturally-occurring human SP-D,
wherein the expressed SP-D polypeptide and the naturally-occurring human SP-D each comprise the same percentage of carbohydrate structures comprising: (i) a glycan with 3 sialic acids, (ii) a monoantennary glycan, (iii) a tetraantennary glycan, (iv) a hybrid-type glycan, or (v) a high mannose-type glycan.
2. The method of claim 1 , wherein the cell is a NM-H9D8 cell.
3. The method of claim 1 , wherein the cell is a NM-H9D8(8B11) cell.
4. The method of claim 1 , wherein the polynucleotide encodes a leader polypeptide selected from a wild type SP-D polypeptide leader sequence, and a wild type T-cell receptor (TCR) polypeptide leader sequence.
5. The method of claim 4 , wherein the leader polypeptide comprises the amino acid sequence of SEQ ID NO:05 or SEQ ID NO:10.
6. The method of claim 1 , wherein the polynucleotide encodes a pre-polypeptide comprising a leader polypeptide and the SP-D polypeptide, the pre-polypeptide having an amino acid sequence comprising SEQ ID NO:04 or SEQ ID NO:09.
7. The method of claim 1 , wherein the SP-D polypeptide comprises a residue at a polymorphic position, wherein the residue is selected from the group consisting of Met11/31, Thr160/180, Ser 270/290, and Ala 286/306.
8. The method of claim 1 , further comprising isolating a population of the expressed SP-D polypeptides, each expressed SP-D polypeptide comprising a complex-type carbohydrate attached at an N-glycosylation site, wherein the population has a glycosylation pattern comprising the following characteristics:
(i) at least 70% of the complex-type carbohydrates include a core fucose;
(ii) at least 10% of the complex-type carbohydrates include at least one sialic acid residue;
(iii) at least 50% of the complex-type carbohydrates include at least a biantennary carbohydrate structure;
(iv) at least 10% of the complex-type carbohydrates include a bisecting N-acetylglucosamine;
(v) less than 10% of the carbohydrates are high-mannose type structures; and
(vi) a detectable amount of α2,6-coupled sialic acid residues.
9. The method of claim 8 , wherein the population has a glycosylation pattern comprising one or more of the following characteristics:
(i) at least 20% of the complex-type carbohydrates include a bisecting N-acetylglucosamine; and
(iii) at least 85% of the complex-type carbohydrates include a core fucose.
10. The method of claim 1 , wherein the polynucleotide encodes a dihydrofolate reductase polypeptide, wherein culturing the cell comprises contacting the cell with an antifolate, and wherein expression of the SP-D polypeptide is increased by increasing the concentration of the antifolate.
11. The method of claim 1 , wherein the cell is cultured in a perfusion bioreactor or in a continuous culture.
12. The method of claim 1 , wherein culturing the cell comprises maintaining a growth medium having a pH 7.2, dissolved oxygen in a range less than 40% and greater than 20%, and temperature at 37° C.
13. The method of claim 1 , wherein the polynucleotide is an expression vector encoding the SP-D polypeptide, wherein the expression vector encodes a leader polypeptide, and a dihydrofolate reductase, wherein the leader polypeptide is selected from a wild type SP-D polypeptide leader sequence or a wild type T-cell receptor (TCR) polypeptide leader sequence.
14. The method of claim 13 , wherein the leader polypeptide comprises SEQ ID NO: 05 or SEQ ID NO: 10.
15. The method of claim 13 , wherein the expression vector encodes a pre-polypeptide comprising the leader polypeptide and the SP-D polypeptide, the pre-polypeptide having an amino acid sequence comprising SEQ ID NO:04 or SEQ ID NO:09.
16. An immortalized human cell comprising an expression vector encoding a leader polypeptide, a human surfactant protein D (SP-D) polypeptide, and a dihydrofolate reductase, wherein the leader polypeptide is selected from a wild type SP-D polypeptide leader sequence or a wild type T-cell receptor (TCR) polypeptide leader sequence,
wherein the cell is selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, NM-H9D8(8B11), and NM-F9, and
wherein the cell is capable of expressing the SP-D polypeptide comprising a glycosylation pattern substantially similar to a glycosylation pattern of a naturally-occurring human SP-D,
wherein the expressed SP-D polypeptide and the naturally-occurring human SP-D each comprise the same percentage of carbohydrate structures comprising: (i) a glycan with 3 sialic acids, (ii) a monoantennary glycan, (iii) a tetraantennary glycan, (iv) a hybrid-type glycan, or (v) a high mannose-type glycan.
17. The cell of claim 16 , wherein the cell is selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, and NM-F9.
18. The cell of claim 16 , wherein the cell is a NM-H9D8 cell.
19. The cell of claim 16 , wherein the cell is a NM-H9D8(8B11) cell.
20. The method of claim 1 , wherein the expressed SP-D polypeptide and the naturally-occurring human SP-D each have an antennarity number within a range from 190 to 215.