AU2772902A - Amino acid modified polypeptides - Google Patents

Amino acid modified polypeptides Download PDF

Info

Publication number
AU2772902A
AU2772902A AU27729/02A AU2772902A AU2772902A AU 2772902 A AU2772902 A AU 2772902A AU 27729/02 A AU27729/02 A AU 27729/02A AU 2772902 A AU2772902 A AU 2772902A AU 2772902 A AU2772902 A AU 2772902A
Authority
AU
Australia
Prior art keywords
cell
amino acid
acid analog
hydroxyproline
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
AU27729/02A
Inventor
Douglas D. Buechter
Kevin Connolly
Elliott Gruskin
Guanghui Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
United States Surgical Corp
Original Assignee
United States Surgical Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by United States Surgical Corp filed Critical United States Surgical Corp
Priority to AU27729/02A priority Critical patent/AU2772902A/en
Publication of AU2772902A publication Critical patent/AU2772902A/en
Abandoned legal-status Critical Current

Links

Landscapes

  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

S&FRef: 531038D1
AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT
ORIGINAL
Name and Address of Applicant: Actual Inventor(s): Address for Service: United States Surgical Corporation 150 Glover Avenue Norwalk Connecticut 06856 United States of America Elliott Gruskin, Douglas D. Buechter, Guanghui Zhang, Kevin Connolly Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Amino Acid Modified Polypeptides Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c AMINO ACID MODIFIED POLYPEPTIDES
BACKGROUND
1. Technical Field Engineered polypeptides having incorporated amino acids which enhance or otherwise modify properties of such polypeptides.
2. Description of Related Art Genetic engineering allows polypeptide production to be transferred from one organism to another. In doing so, a portion of the production apparatus indigenous to an original host is transplanted into a recipient. Frequently, the original host has evolved certain unique processing pathways in association with i 15 polypeptide production which are not contained in or transferred to the recipient.
For example, it is well known that mammalian cells incorporate a complex set of post-translational enzyme systems which impart unique characteristics to protein products of the systems. When a gene encoding a protein normally produced by mammalian cells is transferred into a bacterial or yeast cell, the protein may not be S 20 subjected to such post translational modification and the protein may not function as originally intended.
Normally, the process of polypeptide or protein synthesis in living cells involves transcription of DNA into RNA and translation of RNA into protein.
i* Three forms of RNA are involved in protein synthesis: messenger RNA (mRNA) 25 carries genetic information to ribosomes made of ribosomal RNA (rRNA) while transfer RNA (tRNA) links to free amino acids in the cell pool. Amino acid/tRNA complexes line up next to codons of mRNA, with actual recognition and binding being mediated by tRNA. Cells can contain up to twenty amino acids which are combined and incorporated in sequences of varying permutations into proteins.
Each amino acid is distinguished from the other nineteen amino acids and charged to tRNA by enzymes known as aminoacyl-tRNA synthetases. As a general rule, amino acid/tRNA complexes are quite specific and normally only a molecule with an exact stereochemical configuration is acted upon by a particular aminoacyl-tRNA synthetase.
0 15 o 20 oo o In many living cells some amino acids are taken up from the surrounding environment and some are synthesized within the cell from precursors, which in turn have been assimilated from outside the cell. In certain instances, a cell is auxotrophic, it requires a specific growth substance beyond the minimum required for normal metabolism and reproduction which it must obtain from the surrounding environment. Some auxotrophs depend upon the external environment to supply certain amino acids. This feature allows certain amino acid analogs to be incorporated into proteins produced by auxotrophs by taking advantage of relatively rare exceptions to the above rule regarding stereochemical specificity of aminoacyltRNA synthetases. For example, proline is such an exception, the amino acid activating enzymes responsible for the synthesis of prolyl-tRNA complex are not as specific as others. As a consequence certain proline analogs have been incorporated into bacterial, plant, and animal cell systems. See Tan et al., Proline Analogues Inhibit Human Skin Fibroblast Growth and Collagen Production in Culture, Journal of Investigative Dermatology, 80:261-267(1983).
A method of incorporating unnatural amino acids into proteins is described, in Noren et al., A General Method For Site-Specific Incorporation of Unnatural Amino Acids Into Proteins, Science, Vol. 244, pp. 182-188 (1989) wherein chemically acylated suppressor tRNA is used to insert an amino acid in response to a stop codon substituted for the codon encoding residue of interest. See also, Dougherty et al., Synthesis of a Genetically Engineered Repetitive Polypeptide Containing Periodic Selenomethionine Residues, Macromolecules, Vol. 26, No. 7, pp. 1779-1781 (1993), which describes subjecting an E. coli methionine auxotroph to selenomethionine containing medium and postulates on the basis of experimental data that selenomethionine may completely replace methionine in all proteins produced by the cell.
cis-Hydroxy-L-proline has been used to study its effects on collagen by incorporation into eukaryotic cells such as cultured normal skin fibroblasts (see Tan et al., supra) and tendon cells from chick embryos (see Uitto et al., Procollagen Polypeptides Containing cis-4-Hydroxy-L-proline are Overglycosylated and Secreted as Nonhelical Pro-y-Chains, Archives of Biochemistry and o
A
Biophysics, 185:1:214-221(1978)). However, investigators found that trans-4hydroxyproline would not link with proline specific tRNA of prokaryotic E. coli.
See Papas et al., Analysis of the Amino Acid Binding to the Proline Transfer Ribonucleic Acid Synthetase of Escherichia coli, Journal of Biological Chemistry, 245:7:1588-1595(1970). Another unsuccessful attempt to incorporate trans-4hydroxyproline into prokaryotes is described in Deming et al., In Vitro Incorporation of Proline Analogs into Artificial Proteins, Poly. Mater. Sci. Engin.
Proceed., Vol. 71, p. 673-674 (1994). Deming et al. report surveying the potential for incorporation of certain proline analogs, L-azetidine-2-carboxylic acid, Ly-thiaproline, 3,4-dehydroproline and L-trans-4-hydroxyproline into artificial S 15 proteins expressed in E. coli cells. Only L-azetidine-2-carboxylic acid, L-ythiaproline and 3,4 dehydroproline are reported as being incorporated into proteins in E. coli cells in vivo.
Type I collagen is the most abundant form of the fibrillar, interstitial collagens and is the main component of the extracellular matrix. Collagen S 20 monomers consist of about 1000 amino acid residues in a repeating array of Gly-X- Y triplets. Approximately 35% of the X and Y positions are occupied by proline eand 4-hydroxyproline. Collagen monomers associate into triple helices which consist of one a2 and two al chains. The triple helices associate into fibrils which are oriented into tight bundles. The bundles of collagen fibrils are further organized to form the scaffold for extracellular matrix.
i In mammalian cells, post-translational modification of collagen contributes to its ultimate chemical and physical properties and includes proteolytic digestion of pro-regions, hydroxylation of lysine and proline, and glycosylation of hydroxylated lysine. The proteolytic digestion of collagen involves the cleavage of pro regions from the N and C termini. It is known that hydroxylation of proline is essential for the mechanical properties of collagen. Collagen with low levels of 4hydroxyproline has poor mechanical properties, as highlighted by the sequelae associated with scurvy. 4-hydroxyproline adds stability to the triple helix through hydrogen bonding and through restricting rotation about C-N bonds in the polypeptide backbone. In the absence of a stable structure, naturally occurring cellular enzymes contribute to degrading the collagen polypeptide.
The structural attributes of Type I collagen along with its generally perceived biocompatability make it a desirable surgical implant material. Collagen is purified from bovine skin or tendon and used to fashion a variety of medical devices including hemostats, implantable gels, drug delivery vehicles and bone substitutes. However, when implanted into humans bovine collagen can cause acute and delayed immune responses.
As a consequence, researchers have attempted to produce human recombinant collagen with all of its structural attributes in commercial quantities through genetic engineering. Unfortunately, production of collagen by commercial S 15 mass producers of protein such as E. coli has not been successful. A major problem is the extensive post-translational modification of collagen by enzymes not :present in E. coli. Failure of E. coli cells to provide proline hydroxylation of unhydroxylated collagen proline prevents manufacture of structurally sound collagen in commercial quantities.
SUMMARY
0 A method of incorporating an amino acid analog into a polypeptide produced by a cell is provided which includes providing a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, providing growth media containing at least one amino acid analog selected from the group consisting of 25 trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof and contacting the cell with the growth media wherein the at least one amino acid analog is assimilated into the cell and incorporated into at least one polypeptide.
Also provided is a method of substituting an amino acid analog of an amino acid in a polypeptide produced by a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, which includes providing a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, providing growth media containing at least one amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof and contacting the cell with the growth media wherein the at least one amino acid analog is assimilated into the cell and incorporated as a substitution for at least one naturally occurring amino acid in at least one polypeptide.
A method of controlling the amount of an amino acid analog incorporated into a polypeptide is also provided which includes providing at least a first cell selected from the group consisting of prokaryotic cell and eukaryotic cell, providing a first growth media containing a first predetermined amount of at least one amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3hydroxyproline, cis-4-fluoro-L-proline and combinations thereof and contacting the first cell with the first growth media wherein a first amount of amino acid analog is assimilated into the first cell and incorporated into at least one polypeptide. At least a second cell selected from the group consisting of prokaryotic cell and eukaryotic cell, is also provided along with a second growth media containing a second predetermined amount of an amino acid analog selected from the group consisting i' of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and 20 combinations thereof and the at least second cell is contacted with the second growth media wherein a second amount of amino acid analog is assimilated into the second cell and incorporated into at least one polypeptide.
Also provided is a method of increasing stability of a recombinant polypeptide produced by a cell which includes providing a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, and providing growth media containing an amino acid analog selected from the group consisting of trans- 4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof and contacting the cell with the growth media wherein the amino acid analog is assimilated into the cell and incorporated into a recombinant polypeptide, thereby stabilizing the polypeptide.
A method of increasing uptake of an amino acid analog into a cell and causing formation of an amino acid analog/tRNA complex is also provided which includes providing a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, providing hypertonic growth media containing amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof and contacting the cell with the hypertonic growth media wherein the amino acid analog is assimilated into the cell and incorporated into an amino acid analog/tRNA complex. In any of the other above methods, a hypertonic growth media can optionally be incorporated to increase uptake of an amino acid analog into a cell.
A composition is provided which includes a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, and hypertonic media including an amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof.
C BRIEF DESCRIPTION OF THE DRAWINGS 15 Figure 1 is a plasmid map illustrating pMAL-c2.
Figure 2 is a graphical representation of the concentration of intracellular hydroxyproline based upon concentration of trans-4-hydroxyproline in growth culture over time.
Figures 3 and 3A depict a DNA sequence encoding human Type 1 (al) collagen.
S"Figure 4 is a plasmid map illustrating pHuCol.
Figure 5 depicts a DNA sequence encoding a fragment of human Type 1 (al) collagen.
Figure 6 is a plasmid map illustrating pHuCol-Fl.
S' 25 Figure 7 depicts a DNA sequence encoding a collagen-like peptide.
Figure 8 depicts an amino acid sequence of a collagen-like peptide.
Figure 9 is a plasmid map illustrating pCLP.
Figure 10 depicts a DNA sequence encoding mature bone morphogenic protein.
Figure 11 is a plasmid map illustrating pCBC.
Figure 12 is a graphical representation of the percent incorporation of proline and trans-4-hydroxyproline into maltose binding protein under various conditions.
DETAILED DESCRIPTION OF PREFERRED ENBODIMENTS Prokaryotic cells and eukaryotic cells can unexpectedly be made to assimilate and incorporate trans-4-hydroxyproline into proteins contrary to both Papes et al. and Deming et al., supra. Such assimilation and incorporation is especially useful when the structure and function of a polypeptide depends on post translational hydroxylation of proline not provided by the native protein production system of a recombinant host. Thus, prokaryotic bacteria such as E. coli and eukaryotic cells such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis and Schizosaccharomyces pombe that ordinarily do not hydroxylate proline and additional eukaryotes such as insect cells including lepidopteran cell lines including Spodopterafrugiperda, Trichoplasia ni, Heliothis virescens, Bombyx mori infected with a baculovirus; CHO cells, COS cells and NIH 3T3 cells which fail to adequately produce certain polypeptides whose structure and function depend on such hydroxylation can be made to produce polypeptides having hydroxylated prolines. Incorporation includes adding trans-4-hydroxyproline to a polypeptide, for example, by first changing an amino acid to proline, creating a new proline position that can in turn be substituted with trans-4-hydroxyproline or substituting a naturally occurring proline in a polypeptide with rans-4-hydroxyproline as well.
The process of producing recombinant polypeptides in mass producing organisms is well known. Replicable expression vectors such as plasmids, viruses, cosmids and artificial chromosomes are commonly used to transport genes encoding desired proteins from one host to another. It is contemplated that any known method of cloning a gene, ligating the gene into an expression vector and transforming a host cell with such expression vector can be used in furtherance of the present disclosure.
Not only is incorporation of trans-4-hydroxyproline into polypeptides which depend upon trans-4-hydroxyproline for chemical and physical properties useful in production systems which do not have the appropriate systems for converting proline to trans-4-hydroxyproline, but useful as well in studying the structure and function of polypeptides which do not normally contain trans-4-hydroxyproline. It is contemplated that the following amino acid analogs may also be incorporated in accordance with the present disclosure: trans-4 hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof (hereinafter referred to as the "amino acid analogs"). Use of prokaryotes and eukaryotes is desirable since they allow relatively inexpensive mass production of such polypeptides. It is contemplated that the amino acid analogs can be incorporated into any desired polypeptide. In a preferred embodiment the prokaryotic cells and eukaryotic cells are starved for proline by decreasing or eliminating the amount of proline in growth media prior to addition of an amino acid analog herein.
Expression vectors containing the gene for maltose binding protein (MBP), see Figure 1 illustrating plasmid pMAL-c2, commercially available from New S 15 England Bio-Labs, are transformed into prokaryotes such as E. coli proline auxotrophs or eukaryotes such as S. cerevisiae auxotrophs which depend upon externally supplied proline for protein synthesis and anabolism. Other preferred expression vectors for use in prokaryotes are commercially available plasmids which include pKK-223 (Pharmacia), pTRC (Invitrogen), pGEX (Pharmacia), pET (Novagen) and pQE (Quiagen). Substitution of the amino acid analogs for proline in protein synthesis occurs since prolyl tRNA synthetase is sufficiently promiscuous to allow misacylation of proline tRNA with any one of the amino acid analogs. A sufficient quantity, typically ranging from about .001M to about 0.1M, but more preferably from about .005M to about 0.5M of the amino acid analog(s) is added to the growth medium for the transformed cells to compete with proline in cellular uptake. After sufficient time, generally from about 30 minutes to about 24 hours or more, the amino acid analog(s) is assimilated by the cell and incorporated into protein synthetic pathways. As can be seen from Figure 2, intracellular concentration of trans-4-hydroxyproline increases by increasing the concentration of trans-4-hydroxyproline in the growth media. In a preferred embodiment the prokaryotic cells and/or eukaryotic cells are starved for proline by decreasing or eliminating the amount of proline in growth media prior to addition of an amino acid analog herein.
Expression vectors containing the gene for human Type I (al) collagen (DNA sequence illustrated in Figures 3 and 3A; plasmid map illustrated in Figure 4) are transformed into prokaryotic or eukaryotic proline auxotrophs which depend upon externally supplied proline for protein synthesis and anabolism. As above, substitution of the amino acid analog(s) occurs since prolyl tRNA synthetase is sufficiently promiscuous to allow misacylation of proline tRNA with the amino acid analog(s). The quantity of amino acid analog(s) in media given above is again applicable.
Expression vectors containing DNA encoding fragments of human Type 1 (al) collagen DNA sequence illustrated in Figure 5 and plasmid map illustrated in Figure 6) are transformed into prokaryotic or eukaryotic auxotrophs as above. Likewise, expression vectors containing DNA encoding collagen-like 15 polypeptide DNA sequence illustrated in Figure 7, amino acid sequence illustration in Figure 8 and plasmid map illustrated in Figure 9) can be used to transform prokaryotic or eukaryotic auxotrophs as above. Collagen-like peptides are those which contain at least partial homology with collagen and exhibit similar 2 chemical and physical characteristics to collagen. Thus, collagen-like peptides consist, of repeating arrays of Gly-X-Y triplets in which about 35% of the X and Y positions are occupied by proline and 4-hydroxyproline. Certain preferred collagen fragments and collagen-like peptides in accordance herewith are capable of assembling into an extracellular matrix. In both collagen fragments and collagenlike peptides as described above, substitution with amino acid analog(s) occurs since prolyl tRNA synthetase is sufficiently promiscuous to allow misacyclation of proline tRNA with one or more of the amino acid analog(s). The quantity of amino acid Sanalog(s) given above is again applicable.
It is contemplated that any polypeptide having a collagen, collagen fragment or collagen-like peptide domain can be made to incorporate amino acid analog(s) in accordance with the disclosure herein. Such polypeptides include collagen, a collagen fragment or collagen-like peptide domain and a domain having a region incorporating one or more physiologically active substances such as glycoproteins, proteins, peptides and proteoglycans. Physiologically active substances exert control over or modify existing physiologic functions in living things.
Physiologically active substances include hormones, growth factors, enzymes, 5 20
S..
ligands and receptors. Many active domains of physiologically active substances have been defined and isolated. It is contemplated that polypeptides having a collagen, collagen fragment or collagen-like peptide domain can also have a domain incorporating one or more physiologically active domains which are active fragments of such physiologically active substances. Thus, chimeric proteins are made to incorporate amino acid analog(s) by transforming a prokaryotic proline auxotroph or a eukaryotic proline auxotroph with an appropriate expression vector and contacting the transformed auxotroph with growth media containing at least one of the amino acid analogs. For example, a chimeric collagen/bone morphogenic protein (BMP) construct or various chimeric collagen/growth factor constructs are useful in accordance herein. Such growth factors are well-known and include insulin-like growth factor, transforming growth factor, platelet derived growth factor and the like. Figure 10 illustrates DNA of BMP which can be fused to the 3' terminus of DNA encoding collagen, DNA encoding a collagen fragment or DNA encoding a collagen-like peptide. Figure 11 illustrates a map of plasmid pCBC containing a collagen/BMP construct. In a preferred embodiment, proteins having a collagen, collagen fragment or collagen-like peptide domain assemble to form an extracellular matrix which can be used as a surgical implant.
In another aspect, the amount of amino acid analog(s) transport into a target cell can be regulated by controlling the tonicity of the growth media. A hypertonic growth media increases uptake of trans-4-hydroxyproline into E. coli as illustrated in Figure 12. All known methods of increasing osmolality of growth media are appropriate for use herein including addition of salts such as sodium chloride, KCI, MgCl 2 and the like, and sugars such as sucrose, glucose, maltose, etc. and polymers such as polyethylene glycol (PEG), dextran, cellulose, etc. and amino acids such as glycine. Increasing the osmolality of growth media results in greater intracellular concentration of amino acid analog(s) and a higher degree of complexation of amino acid analog(s) to tRNA. As a consequence, proteins produced by the cell achieve a higher degree of incorporation of amino acid analogs. Figure 12 illustrates percentage of incorporation of proline and hydroxyproline into MBP under isotonic and hypertonic media conditions in comparison to proline in native MBP. Thus, manipulating osmolality, in addition to adjusting concentration of amino acid analog(s) in growth media allows a dual-faceted approach to regulating their uptake into prokaryotic cells and eukaryotic cells as described above and consequent incorporation into target polypeptides.
Any growth media can be used herein including commercially available growth media such as M9 minimal medium (available from Gibco Life Technologies, Inc.), LB medium, NZCYM medium, terrific broth, SOB medium and others that are well known in the art.
Collagen from different tissues can contain different amounts of trans-4hydroxyproline. For example, tissues that require greater strength such as bone contain a higher number of trans-4-hydroxyproline residues than collagen in tissues requiring less strength, skin. The present system provides a method of .adjusting the amount of trans-4-hydroxyproline in collagen, collagen fragments, collagen-like peptides, and chimeric polypeptides having a collagen domain, i collagen fragment domain or collagen-like peptide domain fused to a physiologically S 20 active domain, since by increasing or decreasing the concentration of trans-4hydroxyproline in growth media, the amount of trans-4-hydroxyproline incorporated into such polypeptides is increased or decreased accordingly. The collagen, collagen fragments, collagen-like peptides and above-chimeric peptides can be expressed with predetermined levels of trans-4-hydroxyproline. In this manner physical characteristics of an extracellular matrix can be adjusted based upon requirements of end use.
S. Human collagen, collagen fragments, collagen-like peptides and the above chimeric polypeptides produced by recombinant processes have distinct advantages over collagen and its derivatives obtained from non-human animals. Since the human gene is used, the collagen will not act as a xenograft in the context of a medical implant. Moreover, unlike naturally occurring collagen, the extent of proline hydroxylation can be predetermined. This unprecedented degree of control permits detailed investigation of the contribution of trans-4-hydroxyproline to triple helix stabilization, fibril formation and biological activity. In addition, design of medical implants based upon the desired strength of collagen fibrils is enabled.
-11- The following examples are included for purposes of illustration and are not to be construed as limitations herein.
EXAMPLE 1 Trans-membrane Transport A 5 mL culture ofE. coli strain DH5a (supE44 AlacU169 (4801acZ hsdRl7 recA1 endA1 gyrA96 thi-l relAl) containing a plasmid conferring resistance to ampicillin (pMAL-c2, Fig. 1) was grown in Luria Broth to confluency 16 hours from inoculation). These cells were used to inoculate a 1 L shaker flask containing 500 mL of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 pg/mL ampicillin.supplemented with all amino acids at 20 /g/mL) S 15 which was grown to an AUao of 1.0 (18-20 hours). The culture was divided in half and the cells harvested by centrifugation. The cells from one culture, were resuspended in 250 mL M9 media and those from the other in 250 mL of M9 media containing 0.5M NaCI. The cultures were equilibrated in an air shaker for minutes at 37 oC (225 rpm) and divided into ten 25 mL aliquots. The cultures were S* 20 returned to the shaker and 125 /L of 1M hydroxyproline in distilled H 2 0 was added S. to each tube. At 2, 4, 8, 12, and 20 minutes, 4 culture tubes (2 isotonic, 2 hypertonic) were vacuum filtered onto 1 /nm polycarbonate filters that were immediately placed into 2 mL microfuge tubes containing 1.2 mL of 0.2M NaOH/2% SDS in distilled H 2 0. After overnight lysis, the filters were carefully removed from the tubes, and the supernatant buffer was assayed for hydroxyproline according to the method of Grant, Journal of Clinical Pathology, 17:685 (1964).
The intracellular concentration of trans-4-hydroxyproline versus time is illustrated graphically in Figure 2.
EXAMPLE 2 Effects of Salt Concentration on Transmembrane Transport To determine the effects of salt concentration on transmembrane transport, an approach similar to Example 1 was taken. A 5 mL culture of E. coli strain (supE44 AlacU169 (t801acZ AM15) hsdR17 recAl endAl gyrA96 thi-1 relA 1) containing a plasmid conferring resistance to ampicillin (pMAL-c2, Fig. 1) -12was grown in Luria Broth to confluency 16 hours from inoculation). These cells were used to inoculate a 1 L shaker flask containing 500 mL of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 pg/mL ampicillin supplemented with all amino acids at 20 pg/mL) that was then grown to an AU s of 0.6. The culture was divided into three equal parts, the cells in each collected by centrifugation and resuspended in 150 mL M9 media, 150 mL M9 media containing NaCI, and 150 mL M9 media containing 1.OM NaCI, respectively. The cultures were equilibrated for 20 minutes on a shaker at 370 C (225rpm) and then divided into six 25 mL aliquots. The cultures were returned to the shaker and 125 tL of 1M hydroxyproline in distilled H 2 0 was added to each tube. At 5 and minutes, 9 culture tubes (3 isotonic, 3 x 0.5M NaCI, and 3 x 1.OM NaCI) were vacuum filtered onto 1 Am polycarbonate filters that were immediately placed into 2 mL microfuge tubes containing 1.2 mL of 0.2M NaOH/2% SDS in distilled H 2 0.
After overnight lysis, the filters were removed from the tubes and the supernatant buffer assayed for hydroxyproline according to the method of Grant, supra.
EXAMPLE3 Determination Of Proline Starvation Conditions in E. Coli Proline auxotrophic E. coli strain NM519 (pro) including plasmid pMAL-c which confers ampicillin resistance was grown in M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 microgram/mL ampicillin supplemented with all amino acids at 20 Ag/mL except proline which was supplemented at 12.5 mg/L) to a constant AU6o of 0.53 AU (17 hours postinoculation). Hydroxyproline was added to 0.08M and hydroxyproline-dependent growth was demonstrated by the increase in the ODoo to 0.61 AU over a one hour period.
EXAMPLE 4 Hydroxyproline Incorporation Into Protein in E. coli Under Proline Starvation Conditions Plasmid pMAL-c2 (commercially available from New England Biolabs) -13containing DNA encoding for maltose-binding protein (MBP) was used to transform proline auxotrophic E. coli strain NM519 Two 1 L cultures of transformed NM519 (pro') in M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 g/mL ampicillin supplemented with all amino acids at 20 /g/mL except proline which was supplemented at 12.5 mg/L) were grown to an AU6 Of 0.53 (-17 hours post-inoculation). The cells were harvested by centrifugation, the media in one culture was replaced with an equal volume of M9 media containing 0.08M hydroxyproline and the media in the second culture was replaced with an equal volume of M9 media containing 0.08M hydroxyproline and 0.5M NaCI.
After a one hour equilibration,.the cultures were induced with ImM isopropyl-P-Dthiogalactopyranoside. After growing for an additional 3.25 hours, cells were harvested by centrifugation, resuspended in 10 mL of 10mM Tris-HCI (pH ImM EDTA, 100mM NaCl (TEN buffer), and lysed by freezing and sonication. MBP was purified by passing the lysates over 4 mL amylose resin spin columns, washing 2 the columns with 10 mL of TEN buffer, followed by elution of bound MBP with 2 S 20 mL of TEN buffer containing 10mM maltose. Eluted samples were sealed in ampules under nitrogen with an equal volume of concentrated HCI (11.7M) and S.hydrolysed for 12 hours at 120 oC. After clarification with activated charcoal, hydroxyproline content in the samples was determined by HPLC and the method of Grant, supra. The percent incorporation of trans-4-hydroxyproline compared to proline into MBP is shown graphically in Figure 12.
*EXAMPLE Hydroxyproline Incorporation Into Protein in S. cerevisiae via Integrating Vectors Under Proline Starvation Conditions The procedure described in Example 4 above is performed in yeast using an integrating vector which disrupts the proline biosynthetic pathway. A gene encoding human Type 1 (ac) collagen is inserted into a unique shuttle vector behind the inducible GAL10 promoter. This promoter/gene cassette is flanked by a 5' and 3' terminal sequence derived from a S. cerevisiae proline synthetase gene. The plasmid is linearized by restriction digestion in both the 5' and 3' terminal regions and used to transform a proline-prototrophic S. cerevisiae strain. The transformation mixture is plated onto selectable media and transformants are selected. By homologous recombination and gene disruption, the construct simultaneously forms a stable integration and converts the S. cerevisiae strain into a proline auxotroph. A single transformant is selected and grown at 30 oC in YPD media to an ODo of 2 AU. The culture is centrifuged and the cells resuspended in yeast dropout media supplemented with all amino acids except proline and grown to a constant OD60 indicating proline starvation conditions. 0.08M L-hydroxyproline and 2% galactose is then added. Cultures are grown for an additional 6-48 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. Hydroxyproline-containing human Type 1 collagen is purified by ammonium sulfate fractionation and column chromatography.
EXAMPLE 6 Hydroxyproline Incorporation Into Protein in S. cerevisiae via Non-Integrating Vectors Under Proline Starvation Conditions The procedure described above in Example 4 is performed in a yeast S 20 proline auxotroph using a non-integrating vector. A gene encoding human Type 1 S collagen is inserted behind the inducible GALIO promoter in the YEp24 shuttle vector that contains the selectable Ura marker. The resulting plasmid is transformed into proline auxotrophic S. cerevisiae by spheroplast transformation.
The transformation mixture is plated on selectable media and transformants are 25 selected. A single transformant is grown at 30 oC in YPD media to an OD o of 2 AU. The culture is centrifuged and the cells resuspended in yeast dropout media supplemented with all amino acids except proline and grown to a constant ODo indicating proline starvation conditions. 0.08M L-hydroxyproline and 2% (w/v) galactose is then added. Cultures are grown for an additional 6-48 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. Hydroxyproline-containing human Type 1 (az) collagen is purified by ammonium sulfate fractionation and column chromatography.
EXAMPLE 7 Hydroxyproline Incorporation Into Protein in a Baculovirus Expression System A gene encoding human Type 1 (ca 1) collagen is inserted into the pBacPAK8 baculovirus expression vector behind the AcMNPV polyhedron promoter. This construct is co-transfected into SF9 cells along with linearized AcMNPV DNA by standard calcium phosphate co-precipitation. Transfectants are cultured for 4 days at 27 oC in TNM-FH media supplemented with 10 FBS. The media is harvested and recombinant virus particles are isolated by a plaque assay.
Recombinant virus is used to infect 1 liter of SF9 cells growing in Grace's media minus proline supplemented with 10% FBS and 0.08 M hydroxyproline. After growth at 27 oC for 2-10 days, cells are harvested by centrifugation and lysed by mechanical disruption. Hydroxyproline-containing human Type 1 collagen is purified by ammonium sulfate fractionation and column chromatography.
9 9 EXAMPLE 8 Hydroxyproline Incorporation Into Human Collagen Protein in Escherichia coli Under Proline Starvation Conditions A plasmid (pHuCol, Fig. 2) encoding the gene sequence of human Type I collagen (Figures 3 and 3A) placed behind the isopropyl--Dthiogalactopyranoside (IPTG)-inducible tac promotor and also encoding P-lactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro') by standard heat shock transformation. Transformation cultures are plated on Luria Broth (LB) containing 100 pg/ml ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 ml of LB containing 100 pg/ml ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37 oC, this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 pg/ml ampicillin, supplemented with all amino acids at *20 pg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37 oC, 225 rpm, for 15-20 hours post-inoculation, the 20 optical density at 600 nm is constant at approximately 0.5 OD/ml. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 pg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaCI. Following growth for 1 hour at 37 oC, 225 rpm, IPTG is added to ImM and the cultures allowed to grow for an additional 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. Hydroxyproline-containing collagen is purified by e ammonium sulfate fractionation and column chromatography.
EXAMPLE 9 Hydroxyproline Incorporation Into Fragments of Human Collagen Protein in Escherichia coli Under Proline Starvation Conditions A plasmid (pHuCol-Fl, Figure 6) encoding the gene sequence of the first amino acids of human Type 1 collagen (Figure 5) placed behind the isopropyl- P-D-thiogalactopyranoside (IPTG)-inducible tac promotor and also encoding Plactamase is transformed into Escherichia coli proline auxotrophic strain NM519 (pro-) by standard heat shock transformation. Transformation cultures are plated on -17- Luria Broth (LB) containing 100 pg/ml ampicillin and after overnight growth a single ampicillin-resistant colony is used to inoculate 5 ml of LB containing 100 pg/ml ampicillin. After growth for 10-16 hours with shaking (225 rpm) at 37 oC, this culture is used to inoculate 1 L of M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 100 pg/ml ampicillin, supplemented with all amino acids at 20 pg/mL except proline which is supplemented at 12.5 mg/L) in a 1.5 L shaker flask. After growth at 37 oC, 225 rpm, for 15-20 hours post-inoculation, the optical density at 600 nm is constant at approximately 0.5 OD/ml. The cells are harvested by centrifugation (5000 rpm, 5 minutes), the media decanted, and the cells resuspended in 1 L of M9 minimal media containing 100 pg/ml ampicillin, 0.08M L-hydroxyproline, and 0.5M NaC1. Following growth for 1 hour at 37 oC, 225 rpm, IPTG is added to 1mM and the cultures allowed to grow for an additional .i 5-15 hours. Cells are harvested by centrifugation (5000 rpm, 10 minutes) and lysed by mechanical disruption. The hydroxyproline-containing collagen fragment is purified by ammonium sulfate fractionation and column chromatography.
20 It will be understood that various modifications may be made to the embodiments disclosed herein. For example, it is contemplated that any protein produced by prokaryotes and eukaryotes can be made to incorporate one or more amino acid analogs in accordance with the present disclosure. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in art will envision other modifications within the scope and spirit of the claims appended hereto.
Page(s) 2 2 2 7 are claims pages they appear after the sequence listing SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: GRUSKIN, ELLIOTT A.
BUECHTER, DOUGLAS ZHANG, GUANGHUI CONNOLLY, KEVIN (II) TITLE OF INVENTION: AMINO ACID MODIFIED POLYPEPTIDES (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DILWORTH BARRESE STREET: 333 EARLE OVINGTON BOULEVARD CITY: UNIONDALE STATE: NY COUNTRY: US ZIP: .11553 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk S".o COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 08/655,086 FILING DATE: 03-JUN-1996
CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: NAME: STEEN, JEFFREY S.
REGISTRATION NUMBER: 32,063 REFERENCE/DOCKET NUMBER: 203-1632 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 516-228-8484 TELEFAX: 516-228-8516 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 3181 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA -19- (xi) SEQUENCE DESCRIPTION:.SEQ ID NO:1: o 0 0 0 0 t.*
CAGCTGTCTT
GGTCCCTCTG
CCAAGGCTTC
TCCCCGAGGT
TGGTCGTCCT
AGCTGGCCTC
AGATGCTGGT
TGGTCAGATG
TGCTGGTGCT
CCCCGCTGGT
AGGGCCCCGA
TGCTGGTGCT
TGCCAATGGT
TGGACCCCAG
TCCTGGCAGC
ACCCCCTGGC
TGGCCTGCCC
AGATGGTGTT
CCCCAAAGGA
GGGTCTGACT
CGCCGGTCAA
TGTGATGGGA
AGGTGTTCCC
TCAGGGACCC
CTCCCCCGGA
TGGTGAACAG
GAGAGGTTTC
GGCCAACGG7
CGGTAGCCAG
TCCAGGGCC7I
CAAAGATGGC
ATGGCTATGA
GTCCTCGTGG
CAAGGTCCCC
CCCCCAGGTC
GGTGAGCGTG
CCTGGAATGA
CCTGCTGGTC
GGCCCCCGTG
CGTGGAAATG
CCTCCTGGCT
GGCTCTGP
4
AG
GCTGGCCCTG
GCTCCTGGTA
GGCCCCGGCG
AAAGGAGACA
CCTGCTGGAG
GGACCCCCTG
GCTGGTCCCA
TCTCCTGGTG
GGAAGCCCTG
GATGGTCGCC
TTCCCTGGAC
GGACCCCCTG-
CCTGGCCCTG-
TTCCAGGGTC
GGTGTTCCTC
CCTGGCGAGC
GCTCCCGGC2
GGCGCCCCTC
TGAGAAATCA
TCTCCCTGGC
CTGGTGAGCC
CCCCTGGAAA
GGCCTCCTGG
AGGGACACAG
CTAAGGGTGA
GCCTGCCTGG
ATGGTGCTAC
TCCCTGGTGC
GTCCCCAGGG
CTGGAP.ACCC
TTGCTGGTGC
GCCCTCCTGG
CTGGTGCTAA
AGGAAGGA*J
GCGAGCGTGG
*AGGGTCCCGC
AGCTGGTCC
GCPAGCCCTGC
CCGGACCCCC
CTAAAGGTGC
IGCGCTGTCG(
rCTGGTCCCG( TCCCTGGTC4
;GAGACCTTG(
SGTGGTGTGCa k ACGATGGTGI
GCCTTCAGGI
AccGGAGGAA
CCCCCTGGTG
TGGCGAGCCT
GAATGGAGAT
GCCTCAGGGT
AGGTTTCAGT
GCCTGGCAGC
TGAGAGAGGT
TGGTGCTGCC
TGTTGGTGCT
TGTGCGTGGT
TGGTGCTGAT
TCCTGGCTTC
TCCCAAGGGT
GGGAGAGccT
GCGAGGAGCT
TGGACCTGGT
TGGTGAACG7
TCCCGGTGAA
ITCCTGATGGC
AGGCCCACCI
STGCTGGAGAC
;TCCTGCTGGC
TGGCGAGAGI
STGCTGGTCCI
SCGCCCCTGG(
ki AGGTCCCCC! C: TAAGGGTGA!.
r, AATGCCTGG'
TTTCCGTGCC
CACCTGGTCC
GGAGCTTCAG
GATGGGGPIAG
GCTCGAGGAT
GGTTTGGATG
CCTGGTGAAA
CGCCCTGGAG
GGGCCCCCTG
AAGGGTGAAG
GAGCCTGGCO
GGACAGCCTG
CCTGGTGCCC
AACAGCGGTG
GGCCCTGTTG
CGAGGTGAAC
AGCCGTGGTT
GGTTCTCCTG
,GCTGGTCTGC
AAAACTGGCC
GGTGCCCGTG
ICCCGGCAAGG
AhAGATGGAG i GGTGAACAAG V CCAGGTGAAC:
SCCCTCTGGAC
V! GGTCCTGCTC r GCTGGTGCCC r GAACGTGGT(
TGGCCCCATG
CcAPGGcTTc
GTCCCAT(GG
CTGGAAAAcc
TGCCCGGAAC
GTGCCAAGGG
ATGGAGCTCC
CCCCTGGCCC
GTCCCACCGG
CTGGTCCCCA
CCCCTGGCCC
GTGCTAAAGG
GAGGCCCCTC
AACCTGGTGC
GTGTTCAAGG
CCGGACCCAC
TCCCTGGCGC
GCCCCGCTGG
CTGGTGCCAA
CCCCTGGTCC
GTCAGGCTGG
CTGGAGAGCG
AGGCTGGAGC
GCCCTGCTGG
CAGGCAAACC
CRAGAGGCGA
GACCCCGAGG
SCTGGKGCTCC
;CAGCTGGTCT
;GCTCTCCTGG
CTGGTGCCCC
120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 .102 0 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 AAGGGTGACA GAGGTGATGC GTCCGTGGTC TGACCGGCCC TGGTCCCAAA GGTGCTGATC CATTGGTCCT CCTGGCCCT( TGGTGACAAG GGTGAAAGTG GTCCCAGCGG CCCCGGAGAC CGTGGTGAGC CTGGTCCCCC TGCTGACGGC CAhCCTGGTG, CTAAAGGCGA TGGTCCCCCT GGGCCTGCCG, GACCCGCTGG, TCCTGGAGCC AAAGGTGCTC GCGGCAGCGC TGCTGCTGGC CGAGTCGGTC CTCCTGGCCC TGGTCCTGCT GGCAAAGAAG GCGGCAAAGG TCCTGGTGAA GTTGGTCCCC CTGGTCCCCC TGCTGATGGT CCTGCTGGTG CTCCTGGTAC TGGTGTGGTC GGCCTGCCTG, GTCAGAGAGG CTCTGGTGAA. CCTGGCAAAC AAGGTCCCTC TCCCATGGGC CCCCCTGGAT TGGCTGGACC TGCTGCCGAAk GGTTCCCCTG GACGAGACGG GACCGGCCCC GCTGGACCCC CTGGTGCTCC.
CCCTGCTGGC AAGAGTGGTG ATCGTGGTGA CGGCCCCGCT GGCGCCCGTG GCCCCGCCGG, GACAGGCGAA CAGGGCGACA GAGGCATAAA TCCCCCTGGC CCTCCTGGCT CTCCTGGTGA TGGTCCCCGA GGTCCCCCTG GCTCTGCTGG CCCTGGCCCC ATTGGGCCCC CTGGTCCTCG TCCCCCCGGC CCTCCTGGAC CTCCTGGTCC CAGCTTCCTC CCCCAGCCAC CTCAAGAGAA
CCCTGCTGGT
CGGCCCTGCT
ACCTGGTGAT
ACCCCCTGGC
TGGTCCCCCT
CTCTGGAAAT
TCCCCGTGGT
TGGCCCTGCT
TCCCGGGCCT
AGAGAGAGGC
TGGAGCAAGT
CCCTGGTGAA
TTCTCCTGGC
TGGTGCTCCT
GACTGGTCCT
ACCCCPIAGGC
GGGTCACCGT
ACAAGGTCCC
TGCTCCTGGC
CGGTCGCACT
CCCTGGTCCT
CGCTCACGAT
CCCACTGGAG
GGCTTTGCTG
GCTGGTGCCA
CCCATTGGTA
GGTGCTACTG
GCTGGACCCC
GAGACTGGCC
GGCGAGAAAG
CAAGGTATTG
TTCCCTGGTC
GGTGAACGTG
TCTGGACGTG
GCCAAGGGTG
GGTGCCCCTG
GCTGGTCCCG
CCCCGTGGTG
GGCTTCTCTG
TCTGGAGCCT
AAAGATGGAC
GGTGATGCTG,
CCCAGCGCTG,
GGTGGCCGCT
CTCGTGGTGC
GCCCCCCTGG
A.AGGCGATGC
ATGTTGGTGC
GTITCCCTGG
CTGGCCCTCC
CTGCTGGACG
GATCCCCTGG
CTGGACAGCG
TTCCTGGCCC
GTCCCCCCGG
AGGGGGCTCC
ACCGTGGTGA
GCCCCGTTGG
CCGGTCCCGT
ACAAGGGTGA
GCCTCCAGGG
CTGGTCCTGC
TCAACGGTCT
GTCCTGTTGG,
GTTTCGACTT
ACTACCGGGC
1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 T 3181 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 240 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CAGCTGTCTT ATGGCTATGA TGAGAAATCA ACCGGAGGAA TTTCCGTGCC TGGCCCCATG GGTCCCTCTG GTCCTCGTGG TCTCCCTGGC CCCCCTGGTG CACCTGGTCC CCAAGGCTTC 120 -21- 0 CAAGGTCCCC CTGGTGAGCC TGGCGAGCCT GGAGCTTCAG GTCCCATGGG TCCCCGAGGT CCCCCAGGTC CCCCTGGAAA GAATGGAGAT GATGGGGAAG cTGGAARACC TGGTCGTCCT 240 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH; 100 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) M4OLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGATCCATGG GGCTCGCTGG CCCACCGGGC GAACCGGGTC CGCCAGGCCC GAAAGGTCCG CGTGGCGATA GCGGGCTCCC GGGCGATTCC TAATGGATCC 100 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHAIRACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID 1(0:4: Gly Leu Ala Gly Pro Pro Gly Glu Pro Gly Pro Pro Gly Pro Lys Gly :1 5 10 1 Pro Arg Gly Asp Ser INFORMATION FOR SEQ ID SEQ NCE CHARACTERISTICS: (A)LENGTH: 330 base pairs TYPE: nucleic acid S' RANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CAGCGGGCCA GGAAGAAGAA TAAGAACTGC CGGCGCCACT CGCTCTATGT GGACTTCAGC GATGTGGGCT GGAATGACTG GATTGTGGCC CCACCAGGCT ACCAGGCCTT CTACTGCCAT 120 GGGGACTGCC CCTTTCCACT GGCTGACCAC CTCAACTCAA CCAACCATGC CATTGTGCAG 180 ACCCTGGTCA ATTCTGTCMA TTCCAGTATC CCCAAAGCCT GTTGTGTGCC CACTGAACTG 240 AGTGCCATCT CCATGCTGTA CCTGGATGAG TATGATAAGG TGGTACTGAA AAATTATCAG 300 GAGATGGTAG TAGAGGGATG TGGGTGCCGC 330 -22-

Claims (23)

1. A method of incorporating an amino acid analog into a polypeptide produced by a cell selected from the group consisting of prokaryotic cell and eukaryotic cell comprising: providing a cell selected from the group consisting of a prokaryotic cell and eukaryotic cell; providing growth media containing at least one amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof; and contacting the cell with the growth media wherein the at least one amino acid analog is assimilated into the cell and incorporated into at least one :polypeptide.
2. A method according to claim 1 wherein the cell is a proline auxotroph.
3. A method according to claim 2 wherein the cell is selected from the group consisting of bacterial cell, yeast cell and insect cell.
4. A method according to claim 1 wherein the at least one polypeptide is at least a portion of a collagen molecule. A method according to claim 4 wherein the polypeptide is encoded for by the nucleic acid sequence shown in Figure 3.
6. A method according to claim 4 wherein the polypeptide is a fragment encoded for by the nucleic acid sequence shown in Figure
7. A method according to claim 4 wherein the polypeptide is encoded for by the nucleic acid sequence shown in Figure 7. I I'
8. A method according to claim 4 wherein the at least a portion of a collagen is fused to a physiologically active substance.
9. A method according to claim 8 wherein the physiologically active substance is a BMP. A method according to claim 1 wherein nucleic acid encoding the at least one polypeptide is carried on a replicable expression vector.
11. .A method according to claim 10 wherein the replicable expression vector is a plasmid.
12. A method according to claim 3 wherein the proline auxotroph is selected from the group consisting of E. coli and S. cerevisiae.
13. A method according to claim 1 wherein the at least one polypeptide S. is at least a portion of a maltose binding protein molecule.
14. A method according to claim 1 wherein the growth media is hypertonic. :15. A method according to claim 14 wherein an osmolality increasing agent selected from a group consisting of NaCI, KCI, MgC2 1 sucrose, glucose, maltose, PEG, dextran, cellulose and glycine is added to the growth media.
16. A method according to claim 14 wherein NaCI ranges from about to about 1M.
17. A method according to claim 1 wherein the growth media contains an amount of proline which causes proline starvation of the cell.
18. A method of substituting an amino acid analog in a polypeptide manufactured by a cell selected from the group consisting of prokaryotic cell and eukaryotic cell comprising: providing a cell selected from the group consisting of prokaryotic cell and eukaryotic cell; providing growth media containing at least one amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof; and contacting the cell with the growth media wherein the at least one amino acid analog is assimilated into the cell and incorporated as a substitution for at least one naturally occurring amino acid in at least one polypeptide. 15 19. A method of controlling the amount of an amino acid analog incorporated into a polypeptide comprising: providing a first cell selected from the group consisting of prokaryotic cell and eukaryotic cell comprising: providing a first growth media containing a first predetermined S* 20 amount of at least one amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof; and contacting the cell with the first growth media wherein a first amount of amino acid analog is assimilated into the first cell and incorporated into at least 25 one polypeptide; providing at least a second cell selected from the group consisting of prokaryotic cell and eukaryotic cell; providing a second growth media containing a second predetermined amount of an amino acid analog selected from the group consisting of trans-4- hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof; and contacting the at least second cell with the second growth media wherein a second amount of amino acid analog is assimilated into the second cell and incorporated into at least one polypeptide.
20. A method of increasing stability of a recombinant polypeptide produced by a cell comprising: providing a selected from the group consisting of prokaryotic cell and eukaryotic cell; providing growth media containing an amino acid analog selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4- fluoro-L-proline and combinations thereof; and contacting the cell with the growth media wherein the amino acid analog is assimilated into the cell and incorporated into a recombinant polypeptide, thereby stabilizing the polypeptide.
21. A method of increasing uptake of an amino acid analog into a cell and causing formation of an amino acid analog/tRNA complex comprising: providing a prokaryotic cell selected from the group consisting of prokaryotic cell and eukaryotic cell; providing hypertonic growth media containing an amino acid analog S. 20 selected from the group consisting of trans-4-hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof; and contacting the cell with the hypertonic growth media wherein the amino acid analog is assimilated into the cell and incorporated into an amino acid analog/tRNA complex. 9,
22. A composition comprising a cell selected from group consisting of prokaryotic cell and eukaryotic cell and hypertonic growth media including at least one amino acid analog selected from the group consisting of trans-4-hydroxyproline, cis-4- hydroxyproline, 3-hydroxyproline, cis-4-fluoro-L-proline and combinations thereof wherein the hypertonic growth media increases cell uptake of the at least one amino acid analog.
23. A method of incorporating an amino acid analog into a polypeptide produced by a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, said method substantially as hereinbefore described with reference to any one of the examples. 0t 24. A method of substituting an amino acid analog in a polypeptide manufactured by a cell selected from the group consisting of prokaryotic cell and eukaryotic cell, said S method substantially as hereinbefore described with reference to any one of the examples.
25. A method of controlling the amount of an amino acid analog incorporated into a polypeptide, said method substantially as hereinbefore described with reference to any one of the examples. S26. A method of increasing stability of a recombinant polypeptide produced by a cell, said method substantially as hereinbefore described with reference to any one of the examples.
27. A method of increasing uptake of an amino acid analog into a cell and causing 20 formation of an amino acid analog/tRNA complex, said method substantially as hereinbefore described with reference to any one of the examples.
28. A polypeptide produced by the method according to any one of claims 1 to and 23 to 26.
29. An amino acid analog/tRNA complex prepared by the method according to claim 21 or claim 27. A composition comprising a cell and a hypertonic growth media, said composition substantially as hereinbefore described with reference to any one of the examples. Dated 27 March, 2002 United States Surgical Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBUU]02266.doc:MCN
AU27729/02A 1998-05-20 2002-03-28 Amino acid modified polypeptides Abandoned AU2772902A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU27729/02A AU2772902A (en) 1998-05-20 2002-03-28 Amino acid modified polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU746967 1998-05-20
AU27729/02A AU2772902A (en) 1998-05-20 2002-03-28 Amino acid modified polypeptides

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
AU75813/98A Division AU746967B2 (en) 1996-06-03 1998-05-20 Amino acid modified polypeptides

Publications (1)

Publication Number Publication Date
AU2772902A true AU2772902A (en) 2002-05-16

Family

ID=3716037

Family Applications (1)

Application Number Title Priority Date Filing Date
AU27729/02A Abandoned AU2772902A (en) 1998-05-20 2002-03-28 Amino acid modified polypeptides

Country Status (1)

Country Link
AU (1) AU2772902A (en)

Similar Documents

Publication Publication Date Title
AU746967B2 (en) Amino acid modified polypeptides
Ramshaw et al. Collagens as biomaterials
Báez et al. Recombinant microbial systems for the production of human collagen and gelatin
US6492508B1 (en) Nucleic acids encoding extracellular matrix proteins
US7759090B2 (en) Expression system for producing collagen
RU2001123926A (en) Polypeptide variants with increased heparin binding ability
US20140005356A1 (en) Method for in vivo residue-specific DOPA incorporation into mussel adhesive proteins
Huang et al. Biosynthesis and Applications of Silk‐like and Collagen‐like Proteins
WO2021224316A1 (en) Expression of collagen peptide components in procaryotic systems
KR101304735B1 (en) Methods of producing proteins having triple-helix structure
CA2362496A1 (en) Polypeptide variants with increased heparin-binding ability
CN101787369B (en) DNA sequence of optimized rhBMP (recombinant human bone morphogenetic protein)-2, preparation and purpose of encoding protein thereof
AU2772902A (en) Amino acid modified polypeptides
US20060178506A1 (en) Amino acid modified polypeptides
JP2008099699A (en) Amino acid-modified polypeptide
Klösch et al. Expression and purification of biologically active rat bone morphogenetic protein-4 produced as inclusion bodies in recombinant Escherichia coli
Visser Unravelling the molecular contributions to collagen higher order structure: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
Baumann Functional analysis of collagen galactosyltransferases and the identification of collagens in giant viruses
Myllyharju Recombinant expression of human collagens
Exposito et al. N. 1. Bulleid, KE Kadler

Legal Events

Date Code Title Description
MK1 Application lapsed section 142(2)(a) - no request for examination in relevant period