Unlock instant, AI-driven research and patent intelligence for your innovation.

Advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, method to identify the unique features of aMTDs comprising the same, method to develop the aMTD sequences comprising the same

A technology of macromolecules and transduction domains, applied in the field of intracellular transduction of macromolecules, can solve the problems of low cell permeability, hindering clinical development and application, and poor solubility of recombinant proteins.

Active Publication Date: 2017-08-18
塞里维瑞疗法公司
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many efforts to develop cell-permeable therapeutic proteins by using these reference hydrophobic CPP sequences for further clinical development and application have been hampered by the poor solubility and relatively low cell permeability of recombinant proteins under physiological buffer conditions
Although there is already a consensus that hydrophobic CPP-dependent uptake of protein cargoes is a powerful approach for the development of protein-based biotherapeutics, further improvements are needed to address the critical issue of the influence of non-cargo-specific factors. Cargo-specific factors such as protein aggregation, low solubility / yield, and poor cell / tissue permeability of recombinant CPP fusion proteins

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, method to identify the unique features of aMTDs comprising the same, method to develop the aMTD sequences comprising the same
  • Advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, method to identify the unique features of aMTDs comprising the same, method to develop the aMTD sequences comprising the same
  • Advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, method to identify the unique features of aMTDs comprising the same, method to develop the aMTD sequences comprising the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0305] Example 1. Development of a new advanced macromolecular transduction domain (aMTD)

[0306] The CPPs (MTS / MTM and MTD) derived from the H region of the signal sequence (HRSP) do not share homologous features of common sequence, sequence motif and / or common structure. In the present invention, the goal is to develop improved hydrophobic CPPs that meet the newly identified "key factors" of common sequence and structural motifs formatted with "common functions" to facilitate the integration of proteins with the analyzed CPPs. A similar mechanism translocates across the plasma membrane.

[0307] The structural motif is as follows:

[0308]

[0309] Here, X refers to alanine (A), valine (V), leucine (L) or isoleucine (I); proline (P) can be located at one of U (5 ', 6', 7' or 8'). The remaining U consists of A, V, L or I and the P at 12' is proline.

[0310] In Table 9, general consensus sequence / structural motifs are provided as follows. The amino acid length of the...

Embodiment 2

[0311] Example 2. Construction of expression vectors for recombinant proteins fused with aMTD

[0312] Our newly developed technology allows us to expand our approach for making cell-permeable recombinant proteins. Design expression vectors for histidine-tagged CRA proteins fused to aMTD or r-peptides. To construct expression vectors for recombinant proteins, polymerase chain reaction (PCR) was designed to amplify each designed fusion to CRA aMTD or r-peptide.

[0313] The PCR reaction (100ng genomic DNA, 10pmol of each primer, 0.2mM dNTP mixture, 1× reaction buffer and 2.5U Pfu(+) DNA polymerase (Doctor protein, Korea)) in Nde I (5′) and Sal Restriction site digestion between I (3'), the PCR reaction includes denaturation (95°C), annealing (62°C) and extension (72°C) for 30 seconds each, 35 cycles in total. For the last extension cycle, the PCR reaction was held at 72°C for 5 minutes. Then, they were cloned into the sites of pET-28a(+) vector (Novagen, Madison, WI, USA). ...

Embodiment 3

[0314] Example 3. Inducible expression, purification and preparation of recombinant proteins fused to aMTD and r peptides

[0315] To express the recombinant protein, pET-28a for expression of CRA protein fused to negative control [r-peptide 38 (rP38)], reference hydrophobic CPP (MTM12 and MTD85) and aMTD was transformed in E. coli BL21(DE3) strain ( +) carrier. Cells were grown in LB medium containing kanamycin (50 μg / ml) at 37° C. with vigorous shaking and induced at OD600 = 0.6 by adding 0.7 mM IPTG (Biopure) for 2 hours at 37° C. . The induced recombinant proteins were loaded on a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (InstantBlue, Expedeon, Novexin, UK) (Figure 3).

[0316] E. coli cultures were harvested by centrifugation at 5000 x rpm for 10 minutes and the supernatant discarded. The pellet was resuspended in lysis buffer (50 mM NaH 2 PO 4 , 10mM imidazole, 300mM NaCl, pH 8.0). Cell lysates were sonicated on ice using a sonicator (Sonics and M...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
hydrophilicityaaaaaaaaaa
Login to View More

Abstract

The invention discloses an advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, a method to identify the unique features of aMTDs comprising the same, and a method to develop the aMTD sequences comprising the same. The present invention is to execute macromolecule intracellular transduction technology (MITT) for delivering biologically active macromolecules into the cells; specifically, by exploiting well-enhanced hydrophobic cell penetrating peptide (CPP) - advanced macromolecule transduction domain (aMTD) - to effectively transduce biologically active molecules into the plasma membrane, polynucleotides encoding the same, methods of identifying the same, systems of genetically engineering a biologically active molecule with much enhanced cell-permeability by using the same, methods of importing a biologically active molecule into a cell by using the same, and uses thereof.

Description

technical field [0001] The present invention relates to macromolecular intracellular transduction technology (MITT) for the delivery of biologically active macromolecules to cells; ), efficiently transduces bioactive molecules across the plasma membrane; polynucleotides encoding aMTD; methods of identifying aMTD; by using aMTD, a system for genetically engineering bioactive macromolecules with greatly enhanced cell permeability; by A method for introducing a bioactive macromolecule into cells using aMTD; and uses of aMTD. Background technique [0002] A powerful platform technology for the discovery and development of new drugs is the macromolecular intracellular transduction technology (MITT) made possible by the application of cell-penetrating peptides (CPP), which deliver macromolecular cell permeability. A common problem with small molecules is the potential for off-target drug interactions. Furthermore, a limitation of macromolecules is the fact that proteins and nuc...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00G01N33/68C12N15/11
CPCC07K7/08A61K47/64C07K7/06C07K2319/01C07K2319/10G01N33/5035
Inventor 赵大雄
Owner 塞里维瑞疗法公司
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com