scholarly journals Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

2022 ◽  
Author(s):  
Rachel Kapelner ◽  
Allie Obermeyer
Keyword(s):  
Block Copolymer ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Solution Structure ◽  
Polyelectrolyte Complex ◽  
Fluorescent Protein ◽  
Microphase Separation ◽  
Cellular Delivery ◽  
Protein Charge ◽  
Charge Localization

Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, POEGMA79-b-qP4VP175, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte-complexes to deliver anionic proteins intracellularly as well as the importance of correlating solution structure and desired functional activity.

scholarly journals Topology effects on protein–polymer block copolymer self-assembly

2019 ◽  
Vol 10 (14) ◽  
pp. 1751-1761 ◽  
Author(s):  
Takuya Suguri ◽  
Bradley D. Olsen
Keyword(s):  
Block Copolymer ◽  
Self Assembly ◽  
Fluorescent Protein ◽  
Microphase Separation ◽  
Chain Structure ◽  
Synthetic Polymer ◽  
Red Fluorescent Protein ◽  
Protein Polymer ◽  
Ordering Transitions ◽  
Polymer Block

Bioconjugates made of the model red fluorescent protein mCherry and synthetic polymer blocks show that topology, i.e. the BA, BA2, ABA and ABC chain structure of the block copolymers, where B represents the protein and A and C represent polymers, has a significant effect on ordering transitions and the type and size of nanostructures formed during microphase separation.

Direct protein delivery into intact plant cells using polyhistidine peptides

2021 ◽  
Author(s):  
Yoshino Tanaka ◽  
Yoshihiko Nanasato ◽  
Kousei Omura ◽  
Keita Endoh ◽  
Tsuyoshi Kawano ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fusion Proteins ◽  
Protein Delivery ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Plant Cells ◽  
Adenosine Monophosphate ◽  
Cell Penetrating Peptides ◽  
Intracellular Space ◽  
Intracellular Fluorescence

Abstract Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

scholarly journals Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

scholarly journals ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

2002 ◽  
Vol 13 (9) ◽  
pp. 3078-3095 ◽  
Author(s):  
Annette L. Boman ◽  
Paul D. Salo ◽  
Melissa J. Hauglund ◽  
Nicole L. Strand ◽  
Shelly J. Rensink ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Carboxypeptidase Y ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Adp Ribosylation ◽  
And Function ◽  
Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

Fluorescent and “breathable” CO2 responsive vesicles inspired from green fluorescent protein

2017 ◽  
Vol 8 (40) ◽  
pp. 6283-6288 ◽  
Author(s):  
Lei Xu ◽  
Ning Ren ◽  
Ji Pang ◽  
Hongping Deng ◽  
Xinyuan Zhu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Block Copolymer ◽  
Fluorescent Protein ◽  
Gfp Chromophore ◽  
Green Fluorescent

CO2 responsive fluorescent vesicles from a GFP chromophore labeled block-copolymer could change their size and fluorescence to mimic jellyfish breathing.

scholarly journals Silica-Based Nanoparticles for Protein Encapsulation and Delivery

Nanomaterials ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 886 ◽  
Author(s):  
Filippo Begarani ◽  
Domenico Cassano ◽  
Eleonora Margheritis ◽  
Roberto Marotta ◽  
Francesco Cardarelli ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Fluorescent Protein ◽  
Nanoparticle Synthesis ◽  
Routine Practice ◽  
Aqueous Environment ◽  
Protein Encapsulation ◽  
Test Protein ◽  
Effective Delivery ◽  
Green Fluorescent ◽  
Acidic Organelles

Although conceptually obvious, the effective delivery of proteins in therapeutic applications is far from being a routine practice. The major limitation is the conservation of protein physicochemical identity during the transport to the target site. In this regard, nanoparticle-based systems offer new intriguing possibilities, provided that (i) the harsh and denaturating conditions typically used for nanoparticle synthesis are avoided or mitigated; and (ii) nanoparticle biocompatibility and degradation (for protein release) are optimized. Here, we tackle these issues by starting from a nanoparticle architecture already tested for small chemical compounds. In particular, silica-shielded liposomes are produced and loaded with a test protein (i.e., Green Fluorescent Protein) in an aqueous environment. We demonstrate promising results concerning protein encapsulation, protection during intracellular trafficking and final release triggered by nanoparticle degradations in acidic organelles. We believe this proof of principle may open new applications and developments for targeted and efficient protein delivery.

MICROPHASE SEPARATION AND THE MORPHOLOGY OF BLOCK COPOLYMER MELT FILM

2004 ◽  
Author(s):  
YONGMIN HUANG ◽  
JIANBO TANG ◽  
JIE FENG ◽  
HONGLAI LIU ◽  
YING HU
Keyword(s):  
Block Copolymer ◽  
Microphase Separation

Microphase separation in block copolymer/homopolymer blends: Theory and experiment

1997 ◽  
Vol 106 (8) ◽  
pp. 3318-3328 ◽  
Author(s):  
G. Floudas ◽  
N. Hadjichristidis ◽  
M. Stamm ◽  
A. E. Likhtman ◽  
A. N. Semenov
Keyword(s):  
Block Copolymer ◽  
Microphase Separation ◽  
Homopolymer Blends ◽  
Theory And Experiment

scholarly journals Methods for protein delivery into cells: from current approaches to future perspectives

2020 ◽  
Vol 48 (2) ◽  
pp. 357-365
Author(s):  
Chalmers Chau ◽  
Paolo Actis ◽  
Eric Hewitt
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cellular Damage ◽  
Future Perspectives ◽  
Chemically Modified ◽  
Recent Developments ◽  
Direct Delivery

The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.

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