Proteolistics: a biolistic method for intracellular delivery of proteins

Abstract

In this work, an intracellular protein delivery methodology termed "proteolistics" is described. This method utilizes a biolistic gun apparatus and involves a simple protein/projectile preparation step. The protein to be delivered is mixed with a gold particle microprojectile suspension and is placed onto a gene gun cartridge, where it is dehydrated using either lyophilization or room-temperature air-drying. Subsequent intracellular protein delivery is achieved in plant and mammalian tissues upon bombardment. Because the method does not require modification of delivery agents or cargo biomolecules and involves a simple physical deposition of the protein onto the microprojectiles, there is no restriction on protein type in terms of molecular weight, isoelectric point or tertiary structure. Because the method delivers protein through the widely used gene gun system, it can be readily applied to any tissue or organism amenable to biolistics. A variety of proteins with molecular weight ranging from 24 to 68 kDa and isoelectric point from 4.8 to 10.1 were tested in this work. It is anticipated that this simple and versatile technique will offer biologists a powerful tool for basic research in areas such as understanding of cell and gene functions and for biotechnological applications such as genome editing.

Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

“…The RNPs were delivered into the wheat immature embryos (cv Kenong 199) by particle bombardment according to Ortigosa et al 15. with some modifications.…”

Bioengineering tools for the production of pharmaceuticals: current perspective and future outlook

“…The capped mesoporous silica nanoparticles are then covered with plasmids and distributed by gene gun to N. tabacum cotyledons, in which protein expression is activated upon unsealing and release of the appearance of inducer [87]. Compatibly, Martin-Ortigosa et al [88] have strained to enhance the experimental constraints via gold-plated mesoporous silica nanoparticles and gold nanorods, co-bombardment by microparticles and improved DNA-nano particles addon protocol to carry DNA in onion epidermis cells, maize, and tobacco foliage explants [4]. Such tools can also deliver protein over the wide use of gene gun organization.…”

“…Such tools can also deliver protein over the wide use of gene gun organization. It can be readily useful to any organism or tissue amenable to biolistics tools [88].…”

Biotechnology and synthetic biology approaches for metabolic engineering of bioenergy crops

“…Protoplast‐based genome editing might not be directly applicable for bioenergy crops, which cannot be easily regenerated from protoplasts. However, the same concept may be achieved given that several crop plants can be regenerated from tissue culture and are amenable to biolistic transformation, and that biolistic delivery of protein and nucleic acid into plant cells has been shown to be possible (Martin‐Ortigosa et al ., ; Martin‐Ortigosa and Wang, ).…”