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JP-7856997-B2 - Elastomer protein

JP7856997B2JP 7856997 B2JP7856997 B2JP 7856997B2JP-7856997-B2

Inventors

  • キトルソン ジョシュア
  • リー マイケル
  • ブレスロウアー デイビッド エヌ．
  • ヴィドマイアー ダニエル エム．

Assignees

  • ボルト スレッズ インコーポレイテッド

Dates

Publication Date
20260512
Application Date
20180116
Priority Date
20170113

Claims (20)

  1. A method for producing a composition containing recombinant resilin protein, comprising the steps of: culturing a population of recombinant host cells in a ferment, wherein the recombinant host cells contain a vector comprising a secreted resilin coding sequence, the recombinant host cells secrete recombinant resilin protein encoded by the secreted resilin coding sequence, the recombinant host cells are methylotrope yeast cells, and the recombinant methylotrope yeast host cells produce a secreted fraction of the recombinant resilin, the secreted fraction comprising more than 50% of the total recombinant resilin protein expressed by the recombinant host cells; and purifying the recombinant resilin protein from the ferment.
  2. The method according to claim 1, wherein the recombinant resilin protein is a full-length or truncated native resilin.
  3. The aforementioned native resilin is found in the following insects: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides felis, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, Anopheles darlingi, and Acyrthosiphon The method according to claim 1, comprising an organism selected from the group consisting of *Drosophila pisum*, *Drosophila virilis*, *Drosophila erecta*, *Lutzomyia longipalpis*, *Rhodnius prolixus*, *Solenopsis invicta*, *Culex quinquefasciatus*, *Bactrocera cucurbitae*, and *Trichogramma pretiosum*.
  4. The method according to claim 1, wherein the recombinant resilin protein comprises SEQ ID NO:1.
  5. The method according to claim 1, wherein the recombinant resilin protein comprises SEQ ID NO:4.
  6. The method according to claim 1, wherein the recombinant resilin protein contains an α-conjugation factor secretion signal.
  7. The method according to claim 1, wherein the recombinant resilin protein comprises a FLAG tag.
  8. The method according to claim 1, wherein the vector comprises a plurality of secreted resilin-coding sequences.
  9. The method according to claim 1, wherein the recombinant methanolyzool host cell is a species selected from the group consisting of Pichia (Komagataella phaffii) pastoris, Hansenula polymorpha, Pichia (Scheffersomyces) stipitis, Pichia methanolica, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, and Candida versatilis.
  10. The method according to claim 1, wherein the recombinant methylotrope yeast host cells produce the recombinant resilin at a rate exceeding 2 mg resilin/g dry cell weight/hour.
  11. The method according to claim 1, wherein the recombinant methylotrope yeast host cells secrete the recombinant resilin at a rate exceeding 2 mg resilin/g dry cell weight/hour.
  12. The method according to claim 1, wherein more than 80% of the recombinant resilin is present outside the recombinant methylotrope yeast host cells in the fermented product.
  13. The method according to claim 1, wherein the fermented product contains at least 2 g of recombinant resilin per liter.
  14. The step of purifying the recombinant resilin protein is The method according to claim 1, comprising: centrifuging the fermented product to produce a first pellet fraction and a first supernatant fraction; and isolating the recombinant resilin protein from the first pellet fraction.
  15. The step of purifying the recombinant resilin protein is To produce a solution in which the recombinant resilin protein is soluble, chaotrope is added to the first pellet fraction; The method according to claim 14, further comprising: centrifuging the first pellet fraction containing the chaotrope to produce a second supernatant fraction and a second pellet fraction; and isolating soluble full-length resilin from the second supernatant fraction.
  16. A vector for methylotrope yeast cells comprising multiple secreted resilin coding sequences, wherein the secreted resilin coding sequences encode polypeptides containing α-conjugation factor secretion signals.
  17. The vector according to claim 16, wherein the secreted resilin coding sequence codes for a full-length or truncated native resilin.
  18. The vector according to claim 16, wherein the secreted resilin coding sequence codes for a modified full-length or truncated native resilin.
  19. The vector according to claim 18, wherein the modified resilin comprises the addition, deletion, substitution, or repositioning of an amino acid residue, and the amino acid residue can be crosslinked with another resilin.
  20. The vector according to any one of claims 17 to 19, wherein the full-length or shortened native resilin is derived from an organism selected from the group consisting of Drosophila melanogaster, Drosophila sieboldiana, Aeshna fuscipes, Stinkfly, Cat flea, Bombus ignitus, Confused flour beetle, Honeybee, Parasitic wasp, Body louse, Anopheles gambia, Glossina moorcitans, Atta cephalotes, Anopheles darlingzi, Pea aphid, Drosophila melanogaster, Drosophila kirishimaensis, Sandfly, Giant assassin bug, Red imported fire ant, Culex pipiens, Culex cephalospermum, Trichogramma plethiosum.

Description

Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62/446,230, filed January 13, 2017, which is incorporated herein by reference in its entirety for all purposes. Technical field This disclosure generally relates to elastomer proteins and elastomer protein preparations. Specifically, this disclosure relates to elastomer protein sequences, expression constructs, host cells, and solids. Background: Elastomer proteins are polypeptides that exhibit viscoelastic mechanical properties and include elastin, resilin, abductin, and octopus artery elastomers. Resilin is a particularly interesting elastomer protein because it dissipates very little energy during loading and unloading. Resilin is found in many insects, and its low energy dissipation enables the extraordinary ability of many insect species to jump or flap their wings with extreme efficiency. Due to its unique properties, resilin is an interesting elastomer material that could have many industrial applications. However, resilin exists only in very small amounts in nature and therefore cannot be obtained cost-effectively through insect rearing. Variants of natural resilin and resilin-like proteins (based on resilin sequences) have been recombinantly produced in E. coli (E. coli) cultures by numerous groups. The recombinant proteins are isolated by lysing the cells to extract the recombinantly expressed proteins and using affinity chromatography techniques for purification (Elvin et al., 2005; Charati et al., 2009; McGann et al., 2013). Recombinant resilin and resilin-like proteins are crosslinked targeting tyrosine residues that also form crosslinks in natural resilin (see, e.g., Elvin et al., 2005; Qin et al., 2011). Recombinant resilin is also crosslinked targeting lysine residues (Li et al., 2011) or cysteine residues (McGann et al., 2013). Cross-linked recombinant resilin and resilin-like proteins exhibited mechanical properties similar to natural resilin and possessed elastic energy values higher than 90% (Elvin et al., 2005; Qin et al., 2011; Li et al., 2011). In one study, 70–80 mg of recombinant resilin-like protein was produced per liter of E. coli culture, and the resilin-like protein was purified by Ni-NTA affinity chromatography (Charati et al., 2009). A more efficient expression system was developed, which produced 300–450 mg/L of recombinant resilin-like protein from E. coli host cells (Lyons, et al., 2009). A more efficient method for purifying resilin-like protein from lysed E. coli host cells based on heating after salt precipitation has also been developed (Qin et al., 2011; Lyons et al., 2009). However, an improved system for expressing and purifying elastomer proteins (e.g., resilin and resilin-like proteins) with higher production volumes is desired to provide larger-scale and more efficient protein production. One drawback of recovering expressed proteins by simple precipitation-based purification techniques after cell lysis is that the resulting proteins tend to have low purity due to contamination of the target protein with cellular proteins derived from the lysed cells. Low purity can lead to defects in various products, including low elastic energy. Furthermore, intracellular accumulation of proteins can lead to toxicity and thus a decrease in the efficiency of recombinant elastomer protein production. Therefore, what is needed is an improved method for the expression and purification of recombinant elastomer proteins, including a method for recovering elastomer proteins from the extracellular component. Also needed are improved methods for the expression and purification of recombinant elastomer proteins (e.g., resilins and resilin-like proteins) with greater production efficiency. According to several embodiments, methods for preparing compositions comprising recombinant resilin proteins are provided herein, comprising the steps of: culturing a population of recombinant host cells in a ferment, wherein the recombinant host cells comprise a vector containing a secretory resilin coding sequence, and the recombinant host cells secrete recombinant resilin proteins encoded by the secretory resilin coding sequence; and purifying the recombinant resilin proteins from the ferment. In some embodiments, recombinant resilin proteins are full-length or truncated native resilin proteins. In some aspects, native resilience is found in the following insects: Drosophila sechellia, Acromyrmex echinatior, Aeshna, Haematobia irritans, Ctenocephalides felis, Bombus terrestris, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, Pediculus humanus corporis, Anopheles gambiae, Glossina morsitans, Atta cephalotes, and Anopheles darlingi. The recombinant resilin protein is derived from organisms selected from the group consisting of *Drosophila darlingi*, *Acyrthosiphon pisum*, *Drosophila virilis*, *Drosophila erecta*, *Lutzomyia longipalpis*, *Rhodnius prolixus*, *Solenopsis inv