Yeast strains and methods for producing collagen

Abstract

Strains of yeast genetically engineered to produce increased amounts of non-hydroxylated collagen or hydroxylated collagen are described. An all-in-one vector including the DNA necessary to produce collagen, promotors, and hydroxylating enzymes is also described. Methods for producing non-hydroxylated or hydroxylated collagen are also provided.

Claims

1. A strain of yeast genetically engineered to produce non-hydroxylated collagen wherein the strain comprises a vector comprising an optimized DNA sequence encoding bovine collagen, wherein the DNA sequence is at least 90% identical to SEQ ID NO: 2.

2. The strain of yeast of claim 1 wherein the strain of yeast is selected from the group consisting of Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof.

3. The strain of yeast of claim 1 wherein the vector further comprises a DNA sequence for a promoter for the bovine collagen, wherein the promoter is selected from the group consisting of the AOX1 methanol induced promoter, the Das1-Das2 methanol induced bi-directional promoter, the PHTX1 constitutive bi-directional promoter, a CHO histone promoter, the PGCW14-PGAP1 constitutive bi-directional promoter and combinations thereof.

4. The strain of yeast of claim 1 wherein the vector further comprises a DNA sequence for a selection marker encoding for antibiotic resistance or an auxotrophic marker.

5. The strain of yeast of claim 4 wherein the selection marker encoding for antibiotic resistance encodes for resistance to at least one antibiotic selected from the group consisting of hygromycin, zeocin, geneticin and combinations thereof.

6. The strain of yeast of claim 1 wherein the vector is inserted into the yeast through a method selected from the group consisting of electroporation, chemical transformation, and mating.

7. The strain of yeast of claim 1 wherein the DNA sequence is at least 92.5% identical to SEQ ID NO: 2.

8. The strain of yeast of claim 1 wherein the DNA sequence is at least 95% identical to SEQ ID NO: 2.

9. A method for producing non-hydroxylated bovine collagen comprising growing the strain of yeast according to claim 1 in media for a period of time sufficient to produce the bovine collagen.

10. The method of claim 9 wherein the strain of yeast is selected from the group consisting of Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof.

11. The method of claim 9 wherein the media is selected from the group consisting of buffered glycerol complex media (BMGY), buffered methanol complex media (BMMY), and yeast extract peptone dextrose (YPD).

12. The method of claim 9 wherein the period of time is from 24 hours to 72 hours.

13. The method of claim 12 wherein the yeast is selected from the group consisting of Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof.

14. The method of claim 9 wherein the vector further comprises a DNA sequence for a promoter for the bovine collagen, wherein the promoter comprises PHTX1 constitutive bi-directional promoter or PGCW14-PGAP1 constitutive bi-directional promoter.

15. The method of claim 9 wherein the vector further comprises a DNA sequence for a selection marker encoding for antibiotic resistance or an auxotrophic marker.

16. A strain of yeast genetically engineered to produce hydroxylated bovine collagen wherein the strain comprises a first vector comprising an optimized DNA sequence encoding bovine collagen wherein the DNA sequence is at least 90% identical to SEQ ID NO: 2; and a second vector comprising a DNA sequence encoding P4HA1 and a DNA sequence encoding P4HB; and wherein the vectors have been inserted into the strain of yeast.

17. The strain of yeast of claim 16 wherein the yeast is selected from the group consisting of Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof.

18. The strain of yeast of claim 16 wherein the first vector comprises a DNA sequence for a promoter for the bovine collagen, wherein the promoter is selected from the group consisting of AOX1 methanol induced promoter, Das1-Das2 methanol induced bi-directional promoter, PHTX1 constitutive bi-directional promoter, CHO histone promoter, PGCW14-PGAP1 constitutive bi-directional promoter and combinations thereof.

19. The strain of yeast of claim 16 wherein the second vector comprises a DNA sequence for a promoter for P4HA1 and P4HB, wherein the promoter is PHTX1 constitutive bi-directional promoter or PGCW14-PGAP1 constitutive bi-directional promoter.

20. The strain of yeast of claim 16 wherein the first vector, the second vector, or a combination thereof further comprises a DNA sequence for a selection marker encoding for antibiotic resistance or an auxotrophic marker.

21. The strain of yeast of claim 20 wherein the selection marker encoding for antibiotic resistance encodes for resistance to at least one antibiotic selected from the group consisting of hygromycin, zeocin, geneticin and combinations thereof.

22. The strain of yeast of claim 16 wherein the vector is inserted into the yeast through a method selected from the group consisting of electroporation, chemical transformation, and mating.

23. A method for producing hydroxylated bovine collagen comprising growing the strain of yeast in claim 16 in a media for a period of time sufficient to produce the bovine collagen.

24. The method of claim 23 wherein the strain of yeast is selected from the group consisting of Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof.

25. The method of claim 23 wherein the media is selected from the group consisting of BMGY, BMMY, and YPD.

26. The method of claim 23 wherein the period of time is 24 hours to 72 hours.

27. The method of claim 26 wherein the yeast is selected from the group consisting of Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof.

28. The method of claim 23 wherein the second vector further comprises a DNA sequence for a promoter for the P4HA1 and P4HB, wherein the promoter is PHTX1 constitutive bi-directional promoter or PGCW14-PGAP1 constitutive bi-directional promoter.

29. The method of claim 23 wherein the first vector, the second vector, or a combination thereof further comprises a DNA sequence for a selection marker encoding for antibiotic resistance or an auxotrophic marker.

30. An all-in-one vector comprising: (i) an optimized DNA sequence encoding bovine collage, wherein the DNA sequence is at least 90% identical to SEQ ID NO: 2; (ii) a DNA sequence encoding hydroxylation enzymes comprising P4HA1, P4HB, and combinations thereof, including promoters and terminators; (iii) a DNA sequence for a selection marker, including a promoter and a terminator; (iv) a DNA sequence for origins of replication for yeast and bacteria; (v) DNA sequences with homology to a yeast genome for integration into the genome; and (vi) restriction sites at a position selected from the group consisting of 5′, 3 ′, within the above DNA sequences, and combinations thereof allowing for modular cloning.

31. The all-in-one vector of claim 30 wherein the DNA sequence encoding hydroxylation enzymes comprising P4HA1, P4HB, and combinations thereof comprises PHTX1 constitutive bi-directional promoter or PGCW14-PGAP1 constitutive bi-directional promoter.

32. The all-in-one vector of claim 30 wherein the selection marker encodes for antibiotic resistance or an auxotrophic marker.

33. The all-in-one vector of claim 32 wherein the selection marker encoding for antibiotic resistance encodes for resistance to at least one antibiotic selected from the hygromycin, zeocin, geneticin and combinations thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the vector diagram of MMV 63 which was designed to produce non-hydroxylated collagen.

(2) FIG. 2 shows the vector diagram of MMV77 which was designed to produce non-hydroxylated collagen.

(3) FIG. 3 shows the vector diagram of MMV 129 which was designed to produce non-hydroxylated collagen.

(4) FIG. 4 shows the vector diagram of MMV 130 which was designed to produce non-hydroxylated collagen.

(5) FIG. 5 shows the vector diagram of MMV 78 which was designed to produce hydroxylated collagen.

(6) FIG. 6 shows the vector diagram of MMV 94 which was designed to produce hydroxylated collagen.

(7) FIG. 7 shows the vector diagram of MMV 156 which was designed to produce hydroxylated collagen.

(8) FIG. 8 shows the vector diagram of MMV 191 which was designed to produce hydroxylated collagen.

(9) FIG. 9 shows an all-in-one vector MMV 208 which was designed to produce non-hydroxylated or hydroxylated collagen.

(10) FIG. 10 shows the vector diagram of MMV84

(11) FIG. 11 shows the vector diagram of MMV 150

(12) FIG. 12 shows the vector diagram of MMV140

DETAILED DESCRIPTION OF THE INVENTION

(13) The present invention utilizes yeast to produce collagen. Suitable yeast includes, but are not limited to, those of the genus Arxula, Candida, Komagataella, Pichia, Hansenula, Ogataea, Saccharomyces, Cryptococcus and combinations thereof. The yeast maybe modified or hybridized. Hybridized yeast are mixed breeding of different strains of the same species, different species of the same genus or strains of different genera.

(14) Foreign DNA is inserted into the yeast genome or maintains episomal to produce collagen. The DNA sequence for the collagen is introduced into the yeast via a vector. It is known in the art that modification to the DNA, such as codon optimization, may improve the ability and efficiency of the yeast to translate the DNA. Foreign DNAs are any non-yeast host DNA and include for example, but not limited to, mammalian, Caenorhabditis elegans and bacteria. Suitable mammalian DNA for collagen production in yeast include, but is not limited to, bovine, porcine, kangaroo, alligator, crocodile, elephant, giraffe, zebra, llama, alpaca, lamb, dinosaur and combinations thereof.

(15) The DNA is inserted on a vector, suitable vectors include, but are not limited to, pHTX1-BiDi-P4HA-Pre-P4HB hygro, pHTX1-BiDi-P4HA-PHO1-P4HB hygro, pGCW14-pGAP1-BiDi-P4HA-Prepro-P4HB G418, pGCW14-pGAP1-BiDi-P4HA-PHO1-P4HB Hygro, pDF-Col3A1 optimized Zeocin, pCAT-Col3A1 optimized Zeocin, pDF-Col3A1 optimized Zeocin with AOX1 landing pad, pHTX1-BiDi-P4HA-Pre-Pro-P4HB hygro. The vectors typically include at least one restriction site for linearization of DNA.

(16) It is known in the art that promotors can improve the production of proteins. Promoters are DNA sequences included in the vectors. Suitable promoters for use in the present invention include, but are not limited to, AOX1 methanol induced promoter, PDF de-repressed promoter, PCAT de-repressed promoter, Das1-Das2 methanol induced bi-directional promoter, PHTX1 constitutive Bi-directional promoter, a CHO histone promoter, PGCW14-PGAP1 constitutive Bi-directional promoter and combinations thereof.

(17) A terminator is required at the end of each open reading frame utilized in the vectors incorporated into the yeast. The DNA sequence for the terminator is inserted into the vector.

(18) An origin of replication is necessary to initiate replication. The DNA sequence for the origin of replication is inserted into the vector. The vector may additionally be empisomally maintained.

(19) A DNA sequence containing homology to the yeast genome is necessary and is incorporated into the vector.

(20) Selection markers are used to select yeast cells that have been successfully transformed. The markers sometimes are related to antibiotic resistance. The markers may also be related to the ability to grow with or without certain amino acids (auxotrophic markers). Suitable auxotrophic markers include, but are not limited to ADE, HIS, URA, LEU, LYS, TRP and combinations thereof. A DNA sequence for a selection marker is incorporated into the vector.

(21) Prior to post-translational modification, collagen is non-hydroxylated and degrades in the presence of high pepsin concentration, for example a 1:200 Pepsin may be used to cleave the N-terminal and the C-terminal propeptides of collagen to enable fibrillation, which enables converting collagen to bio fabricated material. Therefore, it is useful to provide hydroxylated collagen. To enable the production of hydroxylated collagen, at least one second protein may be necessary. This second protein is an enzyme known as Prolyl 4-hydroxylase subunit alpha-1, hereafter “P4HA1” and Prolyl 4-hydroxylase subunit beta, hereafter “P4HB”. P4HA1 and P4HB DNA may be inserted into the yeast on a vector to hydroxylate the collagen. Hydroxylated collagen has better thermostability compared to non-hydroxylated collagen and is resistant to high concentration pepsin digestion, for example 1:25 to 1:1, total protein to pepsin ratio.

(22) The engineered yeasts above require multiple vectors and each step in the process of loading vectors into the cell can be very time consuming. Multiple vectors also carry multiple selection markers making it difficult to reuse markers when adding new DNA. We have surprisingly found that an “all-in-one vector” could be constructed with the DNA of the collagen and the DNA of P4HA and P4HB combined on a single vector. Promoters and signal sequences can be modularly added at specified cloning sites. The DNA can be inserted in yeast for hydroxylated or non-hydroxylated collagen depending on the presence or absence of the promotors. The all-in-one vector includes sites for linearization to insert the DNA into the yeast including both for random and site directed integration into the genome.

(23) The term “collagen” refers to any one of the known collagen types, including collagen types I through XX, as well as to any other collagens, whether natural, synthetic, semi-synthetic, or recombinant. It includes all of the collagens, modified collagens and collagen-like proteins described herein. The term also encompasses procollagens and collagen-like proteins or collagenous proteins comprising the motif (Gly-X-Y)n where n is an integer. It encompasses molecules of collagen and collagen-like proteins, trimers of collagen molecules, fibrils of collagen, and fibers of collagen fibrils. It also refers to chemically, enzymatically or recombinantly-modified collagens or collagen-like molecules that can be fibrillated as well as fragments of collagen, collagen-like molecules and collagenous molecules capable of assembling into a nanofiber.

(24) In some embodiments, amino acid residues, such as lysine and proline, in a collagen or collagen-like protein may lack hydroxylation or may have a lesser or greater degree of hydroxylation than a corresponding natural or unmodified collagen or collagen-like protein. In other embodiments, amino acid residues in a collagen or collagen-like protein may lack glycosylation or may have a lesser or greater degree of glycosylation than a corresponding natural or unmodified collagen or collagen-like protein.

(25) The collagen in a collagen composition may homogenously contain a single type of collagen molecule, such as 100% bovine Type I collagen or 100% Type III bovine collagen, or may contain a mixture of different kinds of collagen molecules or collagen-like molecules, such as a mixture of bovine Type I and Type III molecules. Such mixtures may include >0%, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or <100% of the individual collagen or collagen-like protein components. This range includes all intermediate values. For example, a collagen composition may contain 30% Type I collagen and 70% Type III collagen, or may contain 33.3% of Type I collagen, 33.3% of Type II collagen, and 33.3% of Type III collagen, where the percentage of collagen is based on the total mass of collagen in the composition or on the molecular percentages of collagen molecules.

(26) The engineered yeast cells described above can be utilized to produce collagen. In order to do so, the cells are placed in media within a fermentation chamber or vat and fed dissolved oxygen and a source of carbon, under controlled pH conditions for a period of time ranging from twelve hours to 1 week. Suitable media include but are not limited to buffered glycerol complex media (BMGY), buffered methanol complex media (BMMY), and yeast extract peptone dextrose (YPD). Due to the fact that collagen is produced in the yeast cell, in order to isolate the collagen, one must either use a secretory strain of yeast or lyse the yeast cells to release the collagen. The collagen may then be purified through know techniques such as centrifugation, precipitation and the like.

(27) The collagen disclosed herein makes it possible to produce a bio fabricated leather. Methods for converting collagen to bio fabricated leather are taught in patent applications U.S. application Ser. Nos. 15/433,566, 15/433,650, 15/433,632, 15/433,693, 15/433,777, 15/433,675, 15/433,676 and 15/433,877, the disclosures of which are hereby incorporated by reference.

Embodiments of the Invention

(28) The invention includes, but is not limited to genetically engineered strains of yeast and methods for producing collagen.

(29) In a first embodiment, the present invention is directed to a strain of yeast that produces non-hydroxylated collagen including a yeast host; a recombinant DNA of a target protein; and a promoter.

(30) In a second embodiment, the present invention is directed to a strain of yeast that produces hydroxylated collagen including a yeast host; a recombinant DNA of a target protein; a DNA of a second target protein; and at least one promoter.

(31) In a third embodiment, the present invention is directed to an all-in-one vector including a DNA of a target protein; a DNA of a second target protein; and a DNA for at least one promotor. Examples: Gelatin, Collagen I, and to introduce more than one gene.

(32) In a fourth embodiment, the present invention is directed to a method for making collagen.

(33) In a fifth embodiment, the present invention is directed to a method for making hydroxylated collagen.

Detailed Description of Embodiments

(34) As used herein, the term DNA means Deoxyribonucleic Acid.

(35) As used herein, the term titer means the amount of target protein produced.

(36) As used herein, the term bio fabricated leather means the use of biology, engineering and design to create a material with leather-like properties.

(37) As used herein, the term all-in-one vector means a vector that includes all DNAs necessary to produce a desired recombinant protein.

(38) As used herein, the term stable collagen means that after being exposed to high concentration of pepsin at least 75% of the initial concentration of collagen is still present.

(39) The following non-limiting Examples are illustrative of the present invention. The scope of the invention is not limited to the details described in these Examples.

Example 1

Yeast Intended to Produce Recombinant Collagen

(40) Wild type Pichia pastoris from DNA 2.0 was obtained. A MMV 63 (Sequence 9) DNA sequence including a collagen sequence was inserted into wild type Pichia pastoris which generated strain PP28. MMV63 was digested by Pme I and transformed into PP1 (Wild Type Pichia pastoris strain) to generate PP28. The vector MMV63 is shown in FIG. 1.

(41) Native bovine collagen was sequenced (Sequence 1) and the sequence was amplified using the following polymerase chain reaction “PCR” protocol to create a linear DNA sequence:

(42) PfuUltra II Fusion HS DNA Polymerase Protocol

(43) For a 50 ul reaction:

(44) TABLE-US-00001 Component Volume Final Concentration Pfu polymerase 1 ul 10 mM dNTP 1 ul 200 uM  10× Pfu ultra HF reaction 5 ul 1.0x Buffer Primer 1, 5 uM 1 ul 0.1 uM Primer 2, 5 uM 1 ul 0.1 uM DNA Variable 5-30 ng  .sup.  water Total volume made up with water to 50 ul *One of ordinary skill in the art appreciates that multiple primers may be used based on the DNA to be amplified.

(45) Thermocycler Protocol for <10 kb of DNA: 95 C for 2 min, 30 cycles of 95 C for 20 seconds, [primer melting temperature-5 C for 20 seconds 72 C for 15 seconds if <1 kb, otherwise 15 sec per kB, 72 C for 3 min, and 4 C forever.

(46) The linear DNA was cloned following the Gibson Procedure, as follows;

(47) For 2-3 fragments, 0.02-0.5 pmol DNA was used. For 4-6 fragments, 0.2-1.0 pmol DNA was used.

(48) Pmols=(weight in ng)×1000/(base pairs×650 daltons)

(49) Or use NEBioCalculator

(50) Optimized efficiency is 50-100 ng vector with 2-3 fold excess insert (use 5 fold excess if <200 bp). Total volume of PCR fragments should not exceed 20%.

(51) 1. Set up following reaction:

(52) TABLE-US-00002 Recommended Amount of Fragments Used for Assembly 4-6 Fragment Positive 2-3 Fragment Assembly Assembly Control** Total Amount 0.02-0.5 pmols* 0.2-1 pmols* 10 μl of Fragments X μl X μl Gibson Assembly 10 μl 10 μl 10 μl Master Mix (2x) Deionized H.sub.2O 10-X μl 10-X μl 0 Total Volume 20 μl*** 20 μl*** 20 μl 2. Incubate in thermocycler at 50 C for 15 min (2-3 frag) or 60 min (4-6 frag). Store at ice or −20 C before transformation 3. Transform NEB 5-alpha cells with 2 ul of assembly reaction.

(53) The clones were transformed into E. Coli following the procedure below: Thaw 50 ul competent cells (typically 5 alpha) on ice Add 10-100 ng DNA in 2 ul volume Let sit on ice for 5 min Heat shock at 42 C for 10 seconds Let sit on ice for 5 min Meanwhile, prepare tubes or plate with 1 ml Super optimal broth with catabolite repression (“SOC”) liquid medium Transfer competent cells into appropriate tube or well on plate Let shake at 37 C for 1 hour for outgrowth Meanwhile, label plates and place in 37 C incubator to warm up. Spin at 10,000 g for 30 s to concentrate cells at bottom Remove and discard 800 ul of SOC. You should have ˜200 ul leftover Add entire 200 ul to room temperature agar plates. Alternatively, add 10% (20 ul) to plate 1 and 90% (180 ul) to plate 2. Spread on plate using sterile glass beads. Incubate at 37 C overnight

(54) The transformed cells were grown out into colonies and E. Coli Colony PCR was performed according to the procedure below:

(55) GoTaq Green Master Mix Protocol (Taq Polymerase)

(56) For 20 ul reaction:

(57) TABLE-US-00003 Component Volume Final Concentration GoTaq Green MM 2x 10 ul  1x Primer 1, 5 uM 1 ul 0.1 uM Primer 2, 5 uM 1 ul 0.1 uM Colony tooth pick water 8 ul

(58) Thermocycler Protocol:

(59) 95 C for 2 min

(60) 28 cycles of

(61) 95 C for 30 seconds

(62) [primer melting temperature-5 C] for 30 seconds

(63) 72 C for 1 minute per kB

(64) 72 C for 5 min

(65) 4 C forever

(66) To screen the colonies for effectiveness of transformation, agarose DNA Gel procedure was followed as described below:

(67) To make an x % agarose gel (typically 8-12%):

(68) 1. Measure X g agarose to achieve your desired percentage. 1 g=1 ml. For example, to make a 1% gel you measure 1 g agarose into 100 ml Tris base, acetic acid, and ethylene diamine tetraacetic acid buffer (“TAE”) 2. Add agarose to 250 ml flask 3. Bring to 100 ml TAE buffer, or your desired volume 4. Microwave until liquid is clear. For 1% in 100 ml, this takes ˜1 min 30 seconds. 5. Add SYBR Safe DNA stain to 1× (it is at 10,000×, so add your total agarose volume in ml/10 to get total ul to add. For example, if you have 100 ml agarose, add 10 ul) 6. Pour into mold. Remember to add the well slots. 7. Wait 45 min to 1 hr for gel to dry.
To run a gel: 1. Remove the well mold from the dried gel 2. Remove the gel+plastic support (don't take gel off plastic support) and transfer to gel box 3. Pour TAE over gel so that it is completely submerged 4. Load 10-20 ul of ladder. 100 ng should be more than enough to visualize. 5. Load your DNA samples (after they have been mixed with gel loading dye). Gel loading dye is 6× and should be diluted to 1× to load samples (ex: mix 4 ul dye+20 ul DNA and load all 24 ul). DNA PCRed with GoTaq® Green Master Mix already have dye incorporated into the mix, and do not need to have dye added. 100 ng should be more than enough to visualize. Some samples may need to be diluted. 6. Place the wired top on the gel box. The negative (black) should be on the side with the wells. 7. Plug gel box into power supply. Run at 100-120 voltage for 10-30 min.
*Dye migrates opposite from DNA (toward (−) charge). This is why running a gel longer/multiple times is inadvisable and you will not be able to visualize anything. Do not re-use gels. Pour new ones instead. You can also put dye into the buffer itself, which may help with visualization.

(69) In order to purify the vector from E. Coli a DNA prep kit was utilized as described in Zymo Research mini prep kit, following manufacturer's protocol.

(70) Sanger sequencing was performed by Genewiz or Eurofins according to vendor's protocol. The results confirmed that after obtaining transformed clones the DNA sequence is correct.

(71) Large scale DNA preparation was performed using Midi Preparation Kit as described in manufacturer's protocol. Obtained kit from Zymo Research. The results show we generated a significant amount of circular DNA or plasmids.

(72) Plasmids were converted to linear DNA using the Restriction Digestion Guide (from Addgene) as described below: Select restriction enzymes to digest your plasmid. Note: To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence analysis program such as Addgene's Sequence Analyzer. Determine an appropriate reaction buffer by reading the instructions for your enzyme. Note: If you are conducting a double digest (digesting with two enzymes at the same time), you will need to determine the best buffer that works for both of your enzymes. Most companies will have a compatibility chart, such as the double digest finder tool from NEB. If you cannot find a buffer that is appropriate for both of your enzymes, you will need to digest with one enzyme first in the buffer for enzyme 1, re-purify the cut plasmid, and then conduct the second digest in the buffer for enzyme 2. In a 1.5 mL tube combine the following: DNA Restriction Enzyme(s) Buffer (1×) BSA (if recommended by manufacturer) dH.sub.2O up to total volume Note: The amount of DNA that you cut depends on your application. Diagnostic digests typically involve ˜500 ng of DNA, while molecular cloning often requires 1-3 μg of DNA. The total reaction volume usually varies from 10-50 μL depending on application and is largely determined by the volume of DNA to be cut. Note: See Tips and FAQ section below for note on determination of restriction enzyme volume to use. Note: A typical restriction digestion reaction could look like this: 1 μg DNA 1 μL of each Restriction Enzyme 3 μL 10× Buffer 3 μL 10×BSA (if recommended) x μL dH.sub.2O (to bring total volume to 30 μL) Mix gently by pipetting. Incubate tube at appropriate temperature (usually 37° C.) for 1 hour. Always follow the manufacturer's instructions. Note: Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 min to overnight.

(73) The DNA was purified using the Phenol-Chloroform DNA Extraction and Purification procedure described below:

(74) Materials

(75) 1. 3M NaOAc (Sodium Acetate) 2. 100% Ethanol, cold 3. 70% Ethanol, cold 4. Phenol-Chloroform-Isoamyl Alcohol in 25:24:1 ratio
Procedure 1. Add 10% volume of NaOAc to DNA (ex: 50 ul to 500 ul) 2. Add equal volume of phenol-chloroform-isopropanol, careful to take from the bottom/heavier phase; vortex 3. Centrifuge at 12,000 g for 5 min 4. Transfer top phase to a new tube 5. Add 2.5 volumes of cold 100% ethanol, vortex. The liquid should look cloudy if there is a lot of DNA. 6. Put at −80 C for 10 minutes, or on dry ice 7. Centrifuge at max speed for 10 minutes, at 4 C if possible. Remove majority of the supernatant (leave ˜50 ul) 8. Wash with 1 ml cold 70% ethanol, adding wash with no additional mechanical action (do not actively disturb pellet). 9. Centrifuge for 5 min at max speed 10. Remove the majority of the 70% ethanol; leave to air dry for 10-30 min 11. Resuspend in 20-30 ul of water or TE buffer
Notes:
Optimized volumes for microfuge tubes:

(76) 400 ul DNA

(77) 40 ul NaOAc

(78) 440 ul Phenol-Chloroform-Isoamyl Alcohol

(79) Top phase recovered ˜400 ul

(80) Add 1 ml 100% ETOH

(81) The DNA was transformed into yeast cells according to the procedure below: Pichia Electroporation Protocol (Bio-Rad Gene Pulser Xcell™ Total System #1652660)

(82) Pichia strain—WT Pichia from DNA2.0 transformed with P4HA/B co-expression plasmid and selected on Hygro plate (200 ug/ml). Clone #4

(83) 1. Single colony was inoculated in 100 ml YPD medium and grown at 30 degrees overnight with shaking (215 rpm). 2. Next day the culture reaches OD600 ˜3.5 (˜3-5×10.sup.7 cells/OD600). Dilute the culture with fresh YPD to OD600 ˜1.7 and grow for another hour at 30 degree with shaking (215 rpm). 3. Spin down the cells at 3,500 g for 5 min; wash once with water and resuspend in 10 ml 10 mM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT (add fresh), 0.6 M Sorbitol 4. For each transformation, aliquot 8×10.sup.8 cells into 8 ml 10 mM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT, 0.6 M Sorbitol and incubate at room temperature for 30 min. 5. Spin down the cells at 5000 g for 5 mins and wash with ice cold 1.5 ml 1M Sorbitol 3 times and resuspend in 80 ul ice cold 1M Sorbitol 6. Add various amount (about 5 ug) of linearized DNA to the cells and mix by pipetting. 7. Add cells and DNA mixture (80-100 ul) into 0.2 cm cuvette and pulse using Pichia—protocol (1500 v, 25 uF, 200Ω) 8. Immediately transfer the cells into 1 ml mixture of YPD and 1M Sorbitol (1:1) and incubate at 30 degree for >2 hour 9. Plate the cells at different densities.

(84) Inoculate single colonies into 2 mL BMGY media in a 24 deep well plate and grew out for at least 48 hours at 30 degree Celsius with shaking at 900 rpm. The resulting cells were tested for collagen using cell lysis, SDS-page and pepsin assay following the procedure below.

(85) The cells were lysed using the following procedure:

(86) Preparation of 1× lysis buffer. The following recipe is suitable for preparing a combination of 50 samples. 2.5 ml 1 M HEPES; final concentration 50 mM. 438.3 mg NaCl; final concentration 150 mM. 5 ml Glycerol; final concentration 10%. 0.5 ml Triton X-100; final concentration 1%. 42 ml Millipure water. Store buffer at 4° C. for 1 month. Using a Qiagen TissueLyser, lyse Pichia pastoris cells. Speed: 30 hz Time: 15 min (continuous) Centrifuge lysate at 2500 rpm for 15 mins on tabletop centrifuge. Collect about 600 ul of supernatant in a fresh tube or 96 well deep plate. Discard pellet. SDS-Page was performed using the following procedure: Preparation of Buffers and Solutions Mix 50 ml of Pierce™ 20×Tris-Acetate SDS Buffer with 950 ml of Millipure Water to make 1×Tris-Acetate SDS Buffer. Add 1500 ml of 1×Tris-Acetate SDS Buffer to each chamber of the Mini or Midi Gel Tank. SDS-PAGE—Each gel will contain the following: Molecular Weight Markers, Negative Control, Positive Control(s), Samples. Gel Preparation Open plastic casing around gel. Remove well comb from gel. Remove white tape from gel. Place gel into Midi Gel Tank as per manufacturer instructions. Rinse gel wells with 5 ml of 1×Tris-Acetate SDS Buffer 1 ml at a time. Aspirate bubbles and ensure all wells are submerged in 1×Tris-Acetate SDS Buffer. Sample Preparation for Loading SDS-PAGE gel. Thaw samples and controls on ice. Dilute LDS buffer to 2× and add 10% 2-Mercaptoethanol final volume, make up the volume with water. Mix each sample and LDS+2-ME in 1:1 ratio Briefly vortex and centrifuge samples. Incubate all samples at 70° C. for 7 minutes Allow samples to cool to room temperature and briefly centrifuge. Sample Loading Add 20 μL of controls and samples and 10 ul molecular weight standards to each well Electrophoresis for 1 to 4 Midi Gel Tanks Create a one-step program on the PowerEase® 300W.  Step one is 150 V for one hour and 10 minutes. Attach the lid of the Midi Gel Tank to the base as per the manufacturer's instructions. Attach the power cables to the correct outlets on the PowerEase® 300W, making sure the red cable is attached to the red outlet, and the black cable is attached to the black outlet. Repeat as necessary for up to 4 Midi Gel Tanks. Run the one step program. Prepare the gel for transfer. Turn off PowerEase® 300W. Unplug the Midi Gel Tank cables from the PowerEase® 300W. Remove the lid from the Midi Gel Tank. Remove the gel from the Midi Gel Tank. Using the gel knife included with the Midi Gel Tank open the gel's plastic casing by wedging the blade of the knife into the plastic crevice and torqueing the knife. Repeat this motion along the crevice until the plastic case is separated into two. Hold the plastic case with the gel attached to it gel-side down over the Nalgene™ Staining Box containing water and gently press the gel knife into the anode grove to release the gel into the Staining Box. Repeat the following procedure 3 times to wash the gel in Millipore water.  Incubate for 30 seconds Decant the water Coomassie staining: Add 10-20 ml of PageBlue Protein Staining Solution and incubate at room temperature for 60 minutes with gentle agitation on a shaker. Gels may be stained overnight without affecting the background. Discard the staining solution and rinse the gel two times with MilliporeMillipure water. Discard the staining solution and water in a designated biohazard waste container, not down the drain. Add 20 ml of water to destain. For complete destaining, it will take 10-12 hours. For faster destaining, add some methanol to water. Replacing water frequently will enhance destaining.

(87) The pepsin assay was performed with the following procedure: 1. Before pepsin treatment perform BCA assay to obtain the total protein of each sample per Thermo Scientific protocol. Normalize the total protein to the lowest concentration for all samples. (Note: if lowest total protein concentration is less than 0.5 mg/mL do not use that concentration for normalization) 2. Put 100 uL of lysate in a microcentrifuge tube. 3. Create a master mix containing the following: a. 37% HCl (0.6 mL of acid per 100 mL) and b. Pepsin (stock is 1 mg/mL in deionized water, and final addition of pepsin should be at a 1:25 ratio pepsin:total protein (weight:weight). c. Based on step #1 normalization of total protein the amount of pepsin will vary for final addition, adjust using spreadsheet created. 4. After addition of pepsin, mix 3× with pipet and allow the samples to incubate for an hour at room temperature for the pepsin reaction to take place. 5. After an hour, add 1:1 volume of LDS loading buffer containing β-mercaptoethanol to each sample and allow to incubate for 7 minutes at 70° C. (In this situation 100 uL of LDS should be added). 6. Then spin at 14,000 rpm for 1 minute to remove the turbidity. 7. Add 18 uL from the top of sample onto a 3-8% TAE (using TAE buffer) and run gel for 1 hr 10 minutes at 150V. Or after boiling you can immediately place samples into −80° C. until a gel needs to be run.

(88) The results are shown in Table 1 below.

Example 2

Yeast Producing Recombinant Collagen

(89) Example 1 was repeated following the same procedures and protocols with the following changes: A DNA MMV77 (Sequence 10) sequence including a bovine collagen sequence optimized for Pichia expression (Bovine col3A1 optimized, sequence 2) was inserted into the yeast. A pAOX1 promoter (Sequence 3) was used to drive the expression of collagen sequence. A YPD plate containing Zeocin at 500 ug/ml was used to select successful transformants. The resulting strain was PP8. The vector MMV77 is shown in FIG. 2.

(90) Restriction digestion was done using Pme I.

(91) The strains were grown out in BMMY media and tested for collagen. The results are shown in Table 1 below.

Example 3

Yeast Producing Increased Amount of Recombinant Collagen

(92) Example 1 was repeated following the same procedures and protocols with the following changes: A DNA MMV-129 (sequence 11) sequence including a bovine collagen sequence optimized for Pichia expression was inserted into the yeast. A pCAT promoter (Sequence 7) was used to drive the expression of collagen sequence. A YPD plate containing Zeocin at 500 ug/ml was used to select successful transformants. The resulting strain was PP123. MMV129 was digested by Swa I and transformed into PP1 to generate PP123. The vector MMV129 is shown in FIG. 3.

(93) The strains were grown out in BMGY media and tested for collagen. The results are shown in Table 1 below.

Example 4

Yeast Producing Optimal Amount of Recombinant Collagen

(94) Example 1 was repeated following the same procedures and protocols with the following changes:

(95) A DNA MMV-130 (Sequence 12) sequence including a bovine collagen sequence (Sequence 2) optimized for Pichia expression was inserted into the yeast. A pDF promoter (Sequence 6) was used to drive the expression of collagen sequence. An AOX1 landing pad (cut by Pme I, sequence 8) was used to help site specific integration of the vector into the Pichia genome. A YPD plate containing Zeocin at 500 ug/ml was used to select successful transformants. The resulting strain was PP153. MMV130 was digested by Pme I and transformed into PP1 to generate PP153. (Bovine col3A1 optimized, sequence 2). Phenol extraction was not used and PureLink PCR purification kit was used to recover linearized DNA.

(96) The strains were grown out in BMGY media and tested for collagen. The results are shown in Table 1 below.

Example 5

Yeast Intended to Produce Recombinant Hydroxylated Collagen

(97) Example 2 was repeated following the same procedures and protocols with the following changes: One DNA vector, MMV-78 (Sequence 13), containing both bovine P4HA (Sequence 4) and bovine P4HB (sequence 5) sequences were inserted into the yeast. MMV78 was digested by Pme I and transformed into PP1 to generate PP8. Both P4HA and P4HB contain their endogenous signal peptides and are driven by the Das1-Das2 bi-directional promoter (Sequence 25). The DNA was digested by Kpn I and transformed into PP8 to generate PP3. Sequence 2. The vector MMV78 is shown in FIG. 5.

(98) The strains were grown out in BMMY media and tested for collagen and hydroxylation. The results are shown in Table 1 below.

Example 6

Yeast Producing Recombinant Hydroxylated Collagen

(99) Example 2 was repeated following the same procedures and protocols with the following changes: One DNA vector, MMV-78, containing both bovine P4HA and bovine P4HB sequences were inserted into the yeast. Both P4HA and P4HB contain their endogenous signal peptides and are driven by the Das1-Das2 bi-directional promoter. The DNA was digested by Kpn I and transformed into PP8 to generate PP3. Sequence 2.

(100) Another vector, MMV-94 (Sequence 14), containing P4HB driven by pAOX1 promoter was used and was also inserted into the yeast. The endogenous signal peptide of P4HB was replaced by PHO1 signal peptide. The resulting strain was PP38. MMV94 was digested by Avr II and transformed into PP3 to generate PP38. The vector MMV94 is shown in FIG. 6.

(101) The strains were grown out in BMMY media and tested for collagen and hydroxylation. The results are shown in Table 1 below.

Example 7

Yeast Producing Increased Amount of Recombinant Hydroxylated Collagen

(102) Example 4 was repeated following the same procedures and protocols with the following changes: One DNA vector, MMV-156 (Sequence 15), containing both bovine P4HA and bovine P4HB sequences were inserted into the yeast. The P4HA contains its endogenous signal peptides and P4HB signal sequence was replaced with Alpha-factor Pre (Sequence 21) sequence. Both genes were driven by the pHTX1 bi-directional promoter (Sequence 25). MMV156 was digested by Bam HI and transformed into PP153 to generate PP154. Sequence 2. The vector MMV156 is shown in FIG. 7. The strains were grown out in BMGY media and tested for collagen and hydroxylation. The results are shown in Table 1 below.

Example 8

Yeast Producing Optimal Amount Recombinant Hydroxylated Collagen

(103) Example 4 was repeated following the same procedures and protocols with the following changes: One DNA vector, MMV-156, containing both bovine P4HA and bovine P4HB sequences were inserted into the yeast. The P4HA contains its endogenous signal peptides and P4HB signal sequence was replaced with Alpha-factor Pre sequence. Both genes were driven by the pHTX1 bi-directional promoter. The DNA was digested by Swa I and transformed into PP153 to generate PP154. Sequence 2.

(104) Another vector, MMV-191 (Sequence 16), containing both P4HA and P4HB was also inserted into the yeast. The extra copy of P4HA contains its endogenous signal peptide and the signal sequence of the extra copy of P4HB was replaced with Alpha-factor Pre-Pro (Sequence 22) sequence. The extra copies of P4HA and P4HB were driven by the pGCW14-GAP1 bi-directional promoter (Sequence 23). MMV 191 was digested by Bam HI and transformed into PP154 to generate PP268. The vector MMV191 is shown in FIG. 8. The strains were grown out in BMGY media and tested for collagen and hydroxylation. The results are shown in Table 1 below.

Example 9

All-In-One Vector

(105) The methods and procedures of example 1 were utilized to create an all-in-one vector. The All-in-One vector contains DNA of collagen and associated promoter and terminator, the DNA for the enzymes that hydroxylate the collagen and associated promoters and terminators, the DNA for marker expression and associated promoter and terminator, the DNA for origin(s) of replication for bacteria and yeast, and the DNA(s) with homology to the yeast genome for integration. The All-in-one vector contains strategically placed unique restriction sites 5′, 3′, or within the above components. When any modification to collagen expression or other vector components is desired, the DNA for select components can easily be excised out with restriction enzymes and replaced with the user's chosen cloning method. The simplest version of the All-in-one vector (MMV208, Sequence 17) includes all of the above components except promoter(s) for hydroxylase enzymes. Vector MMV208 was made using the following components: AOX homology from MMV84 (Sequence 18), Ribosomal homology from MMV150 (Sequence 19), Bacterial and yeast origins of replication from MMV 140 (Sequence 20), Zeocin marker from MMV140, and Col3A1 from MMV129. Modified versions of P4HA and B and associated terminators were synthesized from Genscript eliminating the following restriction sites: AvrII, NotI, PvuI, PmeI, BamHI, SacII, SwaI, XbaI, SpeI. The vector was transformed into strain PP1.

(106) The strains were grown out in BMGY medium and tested for collagen and hydroxylation. The results are shown in Table 1 below.

(107) TABLE-US-00004 TABLE 1 Example Collagen (g/L) Hydroxylated Collagen (%)  1*  0.05 0 2 0.1 0 3 0.5 0 4 1-1.5 0  5* 0.1 15 6 0.1 35 7 1-1.5 15 8 1-1.5 40-50 9 0.5-1 15-20 *Comparative Examples; in order to quantify collagen, coomassie stained gels were used. A collagen standard curve was used to determine the collagen concentration in the samples. The amount of hydroxylated collagen was estimated by comparing the sample band to a standard band after 1:25 pepsin treatment.

(108) As discussed above, hydroxylated collagen is stable in high concentration of pepsin, therefore its useful not only to have increased amounts of collagen from a fermentation but to also have hydroxylated collagen.

Interpretation of Description

(109) Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof.

(110) Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

(111) Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

(112) Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

(113) The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Incorporation by Reference

(114) All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.