Compositions and methods for protein expression and delivery

A ferritin and assembly technology, applied in the field of compositions and methods for protein expression and delivery, can solve the problems of harmful proteins, harsh conditions, and difficulty in recovering proteins

Pending Publication Date: 2019-07-16
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, methods to provide a permissive folding environment while preventing the accumulation of unfolded intermediates remain a major challenge
[0003] Studies using P22 VLP phage assembled from 420 copies of coat protein and 130-300 copies of other scaffold proteins have shown that recombinant proteins are sequestered within the interior surface (Patterson, D.P. et al. Chem. Commun. (Camb). 49, 10412-10414 (2013)). However, it is more difficult to recover proteins from viral capsids and is harmful to proteins (Comellas-Aragones, M. et al., Nat Nanotechnol.2, 635-639 (2007) )

Method used

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  • Compositions and methods for protein expression and delivery
  • Compositions and methods for protein expression and delivery
  • Compositions and methods for protein expression and delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1: Materials and methods

[0103] Plasmids and Competent Cells

[0104] Cloning was performed using pRSF1b expression vector (Merck) and pBAD / HisB (Life Technologies). Transformations were performed using chemically competent XL1Blue E. coli cells (Simply Science) and BL21(DE3) E. coli cells (Simply Science).

[0105] Reagents and Antibodies

[0106] The following reagents were used: restriction enzymes NcoI, EagI and SpeI (New England BioLabs), ExpresslinkT4 DNA ligase (Life Technologies), Luria-Bertani (LB) agar (Axil Scientific), kanamycin (ThermoFisher), ampicillin ( Axil Scientific), Omega bio-tek Plasmid Mini Kit and Gel Extraction Kit (Simply Science), Site-directed mutagenesis kit (New England BioLabs), isopropyl β-D-1-thiogalactopyranoside, IPTG (Axil Scientific), L-arabinose (Sigma), Tris-HCl pH8.0 (Sigma ), sodium chloride NaCl (Sigma), Triton-X 100 (Sigma), calcium chloride CaCl 2 (Sigma), hemin (Sigma), β-mercaptoethanol (Sigma), imidazole (...

Embodiment 2

[0156] Example 2: Engineering of a Thermally Stable Enclosure (tES)

[0157] The interior of the AfFtn assembly was engineered to maximize cage volume and accommodate the folding requirements of the internalized protein. Examination of the primary sequence and crystal structure of wild-type (WT) AfFtn revealed that subunit interface residues were predominantly hydrophobic and residues in the interior were negatively charged (Johnson, E. et al., Structure. 13, 637- 648 (2005)). Altogether, approximately 25 kDa and 72 negative charges are contributed from the unstructured C-terminus of the WT subunit to the internal volume of the AfFtn assembly. The engineered NE described herein is a C-terminal truncation mutant at residue 164 ( Figures 7A-7F ). The C-terminal truncation did not affect nanocage assembly as confirmed by comparison between the truncation and the size exclusion curves of WT AfFtn. Surprisingly, analytical light scattering studies revealed that removal of the ...

Embodiment 3

[0159] Example 3: Iron uptake properties of tES

[0160] The native AfFtn coat is a physiological iron storage protein; therefore it was investigated whether tES variants also retain iron in our purified preparations or when exposed to in vitro iron loading protocols (Macara IG, et al., The Biochemical journal 126, 151-162 (1972 )). Equimolar amounts (1 μM) of tES(+), tES(-), tES(+ / -) and commercial horse spleen ferritin were compared and no detectable iron core was observed in the purified tES variants ( Figure 10A ) (Johnson, E. et al., Structure. 13, 637-648 (2005)). Then wild-type AfFtn, tES(+), tES(-), tES(+ / -), tES(+)F116H, tES(+)F116H / tES-GFPuv, tES(+) were used in equimolar amounts (1 μM) F116H / tES-HRPC and tES(+)F116H / tES-rLuc tested iron uptake. Wild-type AfFtn showed the highest in vitro iron uptake, whereas the empty tES shell had significant but lower iron accumulation, possibly due to amino acid substitutions near (E128 and E131) ferrous oxidase (Klenk HP et ...

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Abstract

The present invention relates to methods and compositions that provide an environment that promotes enhanced protein expressing and / or folding. More particularly, the present invention provides engineered thermostable ferritin assemblies comprising at least one modified ferritin subunit, wherein the least at least one modified ferritin subunit lacks an unstructured carboxy-terminal sequence that is present in a wildtype ferritin subunit and wherein the engineered assembly is stable at lower salt concentrations that a wildtype ferritin assembly. The invention is exemplified by a ferritin assembly derived from hyperthermophilic Archaeoglobus fulgidus. Also claimed are nucleic acids, compositions, kits and methods of encapsulating a polypeptide, delivering and administering the said engineered ferritin assembly to a subject.

Description

technical field [0001] The present invention relates to methods and compositions that provide an environment that promotes enhanced protein expression and / or folding. More specifically, the invention provides engineered thermostable ferritin assemblies capable of encapsulating polypeptides and nucleic acids. The invention also provides methods and compositions for the delivery of protein therapeutics. Background technique [0002] The path from a nascent polypeptide to a functional protein structure is determined by a balance of factors, some of which act in favor of, and many against, the successful folding of the final product (Hartl, F.U. and Hayer-Hartl, M. ., Nat. Struct. Mol. Biol. 16, 574-581 (2009); Daggett, V. and A. Fersht, A. Nat. Rev. Mol. Cell. Biol. 4, 497-502 (2003)). Natural chaperones prevent the aggregation of unfolded or partially folded intermediates, undergo specialized interactions that favor folding along generative high-energy pathways, and exert ki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47A61K9/51
CPCA61K38/00A61K47/42A61K9/5169C07K14/47A61P35/00C07K14/195A61K39/00C07K14/00
Inventor C·L·德拉姆N·马苏尔卡S·德施潘德G·查克拉博蒂G·瓦莱林特韦德·马韦利
Owner NAT UNIV OF SINGAPORE
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