High molecular weight collagen-like protein polymers

Claim 2

A collagen like polymer of at least about 30 kD, characterized by: being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid and at least 40 number % of said triads comprising at least one proline, wherein the total proline content of said triads is less than 45 number %; and comprising at least two repetitive units of (glyXO) n , wherein X and O symbolize individual amino acids, X and O are the same or different amino acids in each triad and n is at least 4; wherein at least two triads are selected from the group consisting of the triads gly-ala-pro, gly-pro-ala, gly-pro-pro, gly-ala-ser, gly-pro-gly, gly-pro-ser, gly-ala-gln, gly-ser-pro, gly-ser-gln, gly-leu-gln, gly-pro-arg, gly-pro-lys, gly-ala-lys, gly-ala-arg, gly-glu-arg, gly-asp-arg, gly-glu-pro, gly-asp-ala, gly-ala-his and gly-glu-ala.

Claim 3

A collagen like polymer according to claim 1, wherein said polymer comprises at least 15 number % proline of the amino acids and is not more than about 100 kD.

Claim 4

A collagen like polymer according to claim 1, wherein X and O are selected from the group consisting of alanine, isoleucine, valine, leucine, serine, threonine, asparagine, glutamine, lysine, arginine, aspartic acid, glutamic acid, histidine and proline.

Claim 5

A collagen like polymer according to claim 3, wherein the number of triads comprising proline are at least 60 number %.

Claim 6

A collagen like polymer according to claim 1, wherein at least two triads are selected from the group consisting of the triads gly-ala-pro, gly-pro-ala, gly-pro-pro, gly-ala-ser, gly-pro-gly, gly-pro-ser, gly-ala-gln, gly-ser-pro, gly-ser-gln, gly-leu-gln and gly-ala-his.

Claim 7

A collagen like polymer according to claim 1, wherein X and O are selected from the group consisting of alanine, isoleucine, valine, leucine, serine, threonine, asparagine, glutamine, histidine and proline.

Claim 8

A collagen like polymer of at least about 30 kD, characterized by: being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid and at least 40 number % of said triads comprising at least one proline and the proline total content of said triads is less than 45 number %; and comprising at least two repetitive units of the formula α 2 β n α 2 , wherein α has three triads characterized as having glycine as the first amino acid and at least two of the triads having a proline; β has from three to nine triads, wherein at least 30% of the triads have proline; and n is at least 6 and not more than about 36.

Claim 9

A collagen like polymer of at least about 30 kD, characterized by: being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid and at least 40 number % of said triads comprising at least one proline and the proline total content of said triads is less than 45 number %; and comprising at least two repetitive units of the formula α 2 β n α 2 , wherein α has three triads characterized as having glycine as the first amino acid and at least two of the triads having a proline; β has from three to nine triads, wherein at least 30% of the triads have proline; and n is at least 6 and not more than about 36.

Claim 10

A collagen like polymer according to claim 7, wherein α comprises at least one of gly-pro-ala and gly-pro-lys, and β comprises at least two of gly-ala-pro, gly-pro-ala, gly-pro-pro, gly-pro-gly, gly-ala-ser or gly-asp-pro.

Claim 11

A collagen like polymer according to claim 8, wherein β comprises, in one letter amino acid abbreviation, GAPGPAGPP SEQ ID:NO 19, GAPGPAGPPGSRGDPGPP SEQ ID:NO 83, GAQGPAGPGGSRGDPGPP SEQ ID:NO 84, or GAQGPAGPG SEQ ID:NO 85.

Claim 12

A collagen like polymer according to claim 7, wherein said polymer comprises at least four repetitive units.

Claim 13

A DNA sequence encoding a collagen like polymer according to claim 1.

Claim 14

A DNA sequence encoding a collagen like polymer according to claim 7.

Claim 15

A unicellular microorganism comprising a DNA sequence encoding a collagen like polymer of at least about 30 kD and fewer than 45 number % proline said DNA sequence being characterized by: being expressed in said unicellular organism from a construct prepared in vitro; and comprising at least 80 weight % of triads having glycine as the first amino acid and at least 40 number % of said triads comprising at least one proline and the total proline content of said triads is less than 45 number %; and comprising at least two repetitive units of (glyXO) n , wherein X and O are amino acids and are the same or different in each triad and n is at least 4.

Claim 16

A unicellular microorganism comprising a DNA sequence encoding a collagen like polymer of at least about 30 kD and fewer than 45 number % proline said DNA sequence being characterized by: being expressed in said unicellular organism from a construct prepared in vitro; and comprising at least 80 weight % of triads having glycine as the first amino acid and at least 40 number % of said triads comprising at least one proline and the total proline content of said triads is less than 45 number %; and comprising at least two repetitive units of (glyXO) n , wherein X and O are amino acids and are the same or different in each triad and n is at least 4.

Claim 17

A DNA sequence comprising a gene encoding a collagen like polymer of at least about 30 kD, characterized by: capable of being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid; and comprising at least two repetitive units of (glyXO) n , wherein X and O are the same or different in each triad and n is at least 6 where X and O are selected to have from 15 to 45 number percent proline in said polymer; a transcriptional initiation regulatory region functional in a unicellular microorganism 5' of said gene; and a transcriptional termination regulatory region functional in a unicellular microorganism 3' of said gene; wherein said gene is under the transcriptional regulation of said regulatory regions.

Claim 18

A DNA sequence comprising a gene encoding a collagen like polymer of at least about 30 kD, characterized by: capable of being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid; and comprising at least two repetitive units of (glyXO) n , wherein X and O are the same or different in each triad and n is at least 6 where X and O are selected to have from 15 to 45 number percent proline in said polymer; a transcriptional initiation regulatory region functional in a unicellular microorganism 5' of said gene; and a transcriptional termination regulatory region functional in a unicellular microorganism 3' of said gene; wherein said gene is under the transcriptional regulation of said regulatory regions.

Claim 19

A DNA sequence according to claim 14, wherein said gene encodes a collagen like polymer comprising at least one repeat of a sequence of the following formula: ((glyXO).sub.n Ω).sub.m wherein: X and O are any amino acids, except that X and O are selected to have a proline content of the sequence of at least about 10 and less than about 45 number %; Ω has from 0 to 50 amino acids and is other than repetitive glyXO; n is in the range of 4 to 100; and m is at least one.

Claim 20

A DNA sequence according to claim 14, wherein said gene encodes a collagen like polymer of the formula: J.sup.1.sub.r (((glyU.sup.u II.sup.π).sub.p2 (glyX.sup.x O.sup.o).sub.n1 (glyU.sup.u II.sup.π).sub.p2 Ω.sup.Ω).sub.m1)J.sup.2.sub.r wherein: J 1 and J 2 are the same or different and are amino acid sequences of from about 1 to 50 amino acids; X and O are any amino acids except that X and O are selected to have a proline content of the polymer of at least about 30 and less than about 45 number %; Ω is an amino acid sequence of 0 to 50 amino acids; x and o indicate the X and O in each triad may be the same or different; Ω indicates that the amino acid sequence in each Ω may be the same or different; U and II are any amino acids; u and π indicate the same or different amino acids in the individual triads; n 1 is in the range of about 4 to 75; the r's are the same or different and are 0 or 1; p 2 is in the range of 0 to 6; and m 1 is in the range of about 1 to 20.

Claim 21

A method for producing a collagen like polymer of at least about 30 kD, characterized by: being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid; and comprising at least two repetitive units of (glyXO) n , wherein X and O are the same or different in each triad and n is at least 6 where X and O are selected to have from 35 to 45 number percent proline in said polymer; said method comprising: growing a unicellular microorganism comprising a DNA sequence according to claim 11 for sufficient time for said gene to be expressed; and isolating said collagen like polymer.

Claim 22

A formed object comprising a collagen like polymer of at least about 30 kD, characterized by: being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid; and comprising at least two repetitive units of (glyXO) n , wherein X and O are the same or different in each triad and n is at least 4 wherein X and O are selected to have from about 10 to 45 number % proline in said polymer.

Claim 23

A formed object comprising a collagen like polymer of at least about 30 kD, characterized by: being expressed in a unicellular organism from a construct prepared in vitro; comprising at least 80 weight % of triads having glycine as the first amino acid; and comprising at least two repetitive units of (glyXO) n , wherein X and O are the same or different in each triad and n is at least 4 wherein X and O are selected to have from about 10 to 45 number % proline in said polymer.

Claim 24

A formed object according to claim 18, wherein said formed object is a gel, film or fiber.

Claim 25

An antibody composition prepared in response to a collagen like polymer according to claim 1 or fragment of said collagen like polymer.

Claim 26

An antibody composition according to claim 20, wherein said antibody composition is a monoclonal antibody composition.