High molecular weight collagen-like protein polymers

Claim 2

An unnatural collagen-like polymer of at least about 30 kD, characterized by: being capable of being expressed in a unicellular organism from a construct prepared in vitro; comprising a major proportion of amino acids present as 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), wherein X and O are the same or different in each triad and symbolize individual amino acids and n is at least 4.

Claim 3

A collagen-like polymer according to claim 1, wherein at least two triads are triads found in naturally occurring collagens.

Claim 4

A collagen-like polymer according to claim 1, wherein said polymer is not more than about 100 kD and at least 10 number % of the amino acids of said triads are proline.

Claim 5

A collagen-like polymer according to claim 1, wherein X and 0 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 6

A collagen-like polymer according to claim 1, wherein the number of triads comprising proline is at least 60 number %.

Claim 7

An unnatural collagen-like polymer of at least about 30 kD, having a repeating sequence of triads, wherein each of said triads has glycine as the first amino acid and comprising at least one repeat of a sequence of the following formula: ((glyXO).sub.n Ω).sub.m wherein: said polymer is capable of being expressed in a unicellular organism from a construct prepared in vitro; X and O are any amino acids, except that X and O are selected to provide a total proline content of the triads of at least about 10 and less than about 45 % by number; Ω has from 0 to 50 amino acids and is other than a repetitive glyXO motif; n is in the rage of 4 to 1 00; and m is at least one.

Claim 8

An unnatural collagen-like polymer of at least about 30 kD, having a repeating sequence of triads, wherein each of said triads has glycine as the first amino acid and comprising at least one repeat of a sequence of the following formula: ((glyXO).sub.n Ω).sub.m wherein: said polymer is capable of being expressed in a unicellular organism from a construct prepared in vitro; X and O are any amino acids, except that X and O are selected to provide a total proline content of the triads of at least about 10 and less than about 45 % by number; Ω has from 0 to 50 amino acids and is other than a repetitive glyXO motif; n is in the rage of 4 to 1 00; and m is at least one.

Claim 9

A collagen-like polymer according to claim 6, wherein X and 0 are selected from the group consisting of alanine, isoleucine, leucine, valine, serine, threonine, asparagine, glutamine, lysine, arginine, aspartic acid, glutamic acid, histidine and proline.

Claim 10

A collagen-like polymer according to claim 6, wherein at least one of said triads comprises alanine.

Claim 11

A collagen-like polymer according to claim 6, wherein at least one glyXO consists of gly-ala-pro, gly-ala-his, gly-pro-ala, gly-pro-pro, gly-ala-ser, or gly-asp-pro.

Claim 12

A collagen-like polymer according to claim 8, wherein at least about 40 number % of said triads comprise proline.

Claim 13

An unnatural collagen-like polymer of at least about 30 kD, wherein the total proline content of said polymer is less than 45 number %, of the formula: J.sup.1.sub.r ((((glyU.sup.u π.sup.II).sub.p2 (glyX.sup.xol O.sup.o).sub.n1 (glyU.sup.u π.sup.II).sub.p2)Ω.sub.1).sub.m1)J.sup.2, wherein: said polymer is capable of being expressed in a unicellular organism from a construct prepared in vitro; 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 provide a polymer having a proline content of less than about 45 number %; Ω is an amino acid sequence of 0 to 50 amino acids; x and o indicate the X and 0 in each triad may be the same or different; n indicates that the amino acid sequence in each Q may be the same or different; U and π are any amino acids; u and π indicate the same or different amino acids in the individual triads; nphu 1 is in the range of about 4 to 75; the r's are the same or different and are 0 or 1; p2 is in the range of 0 to 6; and m 1 is in the range of about 1 to 20.

Claim 14

An unnatural collagen-like polymer of at least about 30 kD, wherein the total proline content of said polymer is less than 45 number %, of the formula: J.sup.1.sub.r ((((glyU.sup.u π.sup.II).sub.p2 (glyX.sup.xol O.sup.o).sub.n1 (glyU.sup.u π.sup.II).sub.p2)Ω.sub.1).sub.m1)J.sup.2, wherein: said polymer is capable of being expressed in a unicellular organism from a construct prepared in vitro; 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 provide a polymer having a proline content of less than about 45 number %; Ω is an amino acid sequence of 0 to 50 amino acids; x and o indicate the X and 0 in each triad may be the same or different; n indicates that the amino acid sequence in each Q may be the same or different; U and π are any amino acids; u and π indicate the same or different amino acids in the individual triads; nphu 1 is in the range of about 4 to 75; the r's are the same or different and are 0 or 1; p2 is in the range of 0 to 6; and m 1 is in the range of about 1 to 20.

Claim 15

A collagen-like polymer according to claim 11, wherein at least one triad consists of gly-ala-pro, gly-pro-ala, gly-pro-pro, gly-ala-ser, gly-pro-ser, gly-ala-gln, gly-ser-pro, gly-pro-arg, gly-ala-lys, gly-ala-arg, gly-glu-arg, gly-asp-arg, gly-glu-pro, gly-asp-ala or gly-glu-ala.

Claim 16

A collagen like polymer according to claim 11, wherein said polymer comprises at least 40 number % of triads comprising proline.

Claim 17

A collagen-like polymer according to claim 11, wherein U and II are selected from the group consisting of alanine, isoleucine, leucine, valine, serine, threonine, asparagine, glutamine, lysine, arginine, aspartic acid, glutamic acid, histidine and proline.

Claim 18

A collagen-like polymer according to claim 12, wherein the number of triads comprising proline is at least 40 number %.

Claim 19

An unnatural collagen-like polymer of at least about 30 kD, characterized by: being capable of being expressed in a unicellular organism from a construct prepared in vitro; comprising a major proportion of amino acids present as 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 the formula α 2 β 3 α 2 , wherein α has three triads characterized as having glycine as the first amino acid and at least two of the three triads having proline; β has from three to nine triads, wherein at least 30% of the triads have proline; and e is at least 6 and not more than about 36.

Claim 20

An unnatural collagen-like polymer of at least about 30 kD, characterized by: being capable of being expressed in a unicellular organism from a construct prepared in vitro; comprising a major proportion of amino acids present as 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 the formula α 2 β 3 α 2 , wherein α has three triads characterized as having glycine as the first amino acid and at least two of the three triads having proline; β has from three to nine triads, wherein at least 30% of the triads have proline; and e is at least 6 and not more than about 36.

Claim 21

A collagen-like polymer according to claim 16, wherein a 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 22

A collagen-like polymer according to claim 17, wherein β comprises GAPGPAGPP (SEQ ID NO: 133), GAPGPAGPPGSRGDPGPP (SEQ ID NO: 134), GAQGPAGPGGSRGDPGPP (SEQ ID NO: 135), or GAQGPAGPG (SEQ ID NO: 10).

Claim 23

A collagen like polymer according to claim 16, wherein said polymer comprises at least four repetitive units.

Claim 24

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

Claim 25

A DNA sequence encoding a collagen like polymer according to claim 6.

Claim 26

A DNA sequence encoding a collagen like polymer according to claim 11.

Claim 27

A DNA sequence encoding a collagen like polymer according to claim 16.

Claim 28

A unicellular microorganism comprising a DNA sequence encoding an unnatural collagen-like polymer of at least about 30 kD, characterized by comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4.

Claim 29

A unicellular microorganism comprising a DNA sequence encoding an unnatural collagen-like polymer of at least about 30 kD, characterized by comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4.

Claim 30

A DNA sequence comprising: a gene encoding an unnatural collagen-like polymer of at least about 30 kD, characterized by comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4; 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 and wherein said gene is capable of being expressed in a unicellular microorganism from a construct preDared in vitro to provide said collagen-like polymer.

Claim 31

A DNA sequence comprising: a gene encoding an unnatural collagen-like polymer of at least about 30 kD, characterized by comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4; 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 and wherein said gene is capable of being expressed in a unicellular microorganism from a construct preDared in vitro to provide said collagen-like polymer.

Claim 32

A DNA sequence according to claim 25, wherein said gene encodes an unnatural collagen-like polymer comprising at least one repeat of a sequence of the following formula: ((glyXO).sub.n Q).sub.m wherein: X and O are any amino acids, except that X and O are selected to provide for a polymer of less than about 45 number % proline; Ω has from 0 to 50 amino acids and is other than a repetitive glyXO motif; n is in the range of 4 to 100; and m is at least one.

Claim 33

A DNA sequence according to claim 25, wherein said gene encodes a collagen like polymer of the formula: J.sup.1, ((((glyU.sup.u π.sup.n).sub.p2 (glyX.sup.x O.sup.o).sub.n1 (glyU.sup.u π.sup.n).sub.p2)Ω.sub.1).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 provide a polymer having a total proline content of 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; x indicates that the amino acid sequence in each Ω may be the same or different; U and II are any amino acids; u and II 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 34

A method for producing an unnatural collagen-like polymer of at least about 30 kD, characterized by comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4; said method comprising: growing a unicellular microorganism according to claim 24 for sufficient time for said gene to be expressed; and isolating said collagen like polymer.

Claim 35

A formed object comprising an unnatural collagen-like polymer of at least about 30 kD, characterized by: comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4, wherein said collagen-like polymer is capable of being exDressed in a unicellular organism from a construct Drepared in vitro.

Claim 36

A formed object comprising an unnatural collagen-like polymer of at least about 30 kD, characterized by: comprising a major proportion of amino acids present as 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 are the same or different in each triad and symbolize individual amino acids and n is at least 4, wherein said collagen-like polymer is capable of being exDressed in a unicellular organism from a construct Drepared in vitro.

Claim 37

A formed object according to claim 29, wherein said formed object is a gel, film or fiber.

Claim 38

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

Claim 39

An antibody composition according to claim 31, wherein said antibody composition is a monoclonal antibody composition.