Non-activated polypeptides having a function of tissue regeneration and method for preparing the same
View Patent ↗Non-activated tissue-regeneration polypeptides (TRPs) and their preparation methods are disclosed. The TRPs include: a protein transduction domain (PTD) making the polypeptides to permeate a cell membrane without cell membrane receptors; a furin activation domain (FAD) which has at least one proprotein convertase cleavage site and which can be cleaved by the proprotein convertase and activate a tissue regeneration domain (TRD) in cells; and a tissue regeneration domain (TRD) which can be activated by the proprotein convertase cleavage of the FAD to stimulate the growth or formation of tissues or to induce the regeneration of tissues. The TRPs can be mass-produced by cultured bacteria, such as recombinant E. coli , are in a non-activated state before in vivo administration, and their separation, purification, handling, storage and administration are simple and convenient. The in vivo administration of the TRPs is useful to stimulate the formation or regeneration of tissues, such as bones or cartilages, or to improve the fibrosis and cirrhosis of organs, such as kidneys, liver, lungs and heart by pharmacological mechanisms completely different from those of prior rhBMPs or TGF-β proteins.
1. A method for preparing non-activated tissue-regeneration polypeptide (TRP), comprising the steps of:
(a) culturing transformed bacteria to express [PTD-FAD-TRD] a polypeptide in a culture broth, wherein the transformed bacteria are bacteria transformed with a recombinant bacterial expression vector comprising a furin activation domain (FAD)-encoding base sequence upstream of the 5′ region of tissue regeneration domain (TRD)-encoding DNA, a protein transduction domain (PTD) base sequence, a base sequence for tagging, and at least four histidine-encoding base sequences for separation and purification, wherein the PTD is a TAT sequence selected from the group consisting of SEQ ID NOs: 27 and 43, the TRD is an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 6 and the FAD is an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 19; and
(b) centrifuging the culture broth;
(c) removing the secondary or tertiary structure of the polypeptide or converting the secondary or tertiary structure to primary linear structure by the addition of a urea solution into the supernatant and cell pellet to generate a urea, supernatant, cell pellet mixture; and
(d) separating from the mixture and purifying a non-activated polypeptide.
2. The method for preparing non-activated TRP according to claim 1 , wherein the purification step comprises binding the polypeptide to nickel-titanium beads, washing the beads with the urea solution, and then eluting the beads with imidazole and a salt-containing buffer solution.
3. The method for preparing non-activated TRP according to claim 1 , wherein the TRD is SEQ ID NO: and the FAD is SEQ ID NO: 14.
4. The method for preparing non-activated TRP according to claim 1 , wherein the TRD is SEQ ID NO: 6 and the FAD is SEQ ID NO: 19.
5. A recombinant vector for expression in bacteria, comprising a furin activation domain (FAD)-encoding base sequence upstream of the 5′ region of tissue regeneration domain (TRD)-encoding DNA, a protein transduction domain (PTD) base sequence, a base sequence for tagging, and at least four histidine-encoding base sequences for separation and purification,
wherein the TRD is an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 6;
wherein the FAD is an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 19; and
wherein the PTD is a TAT sequence, selected from the group consisting of SEQ ID NOs: 27 and 43.