Synthesis of Glycopolymer Containing Cell-Penetrating Peptides as Inducers of Recombinant Protein Expression under the Control of Lactose Operator/Repressor Systems

Abstract

We recently reported on newly synthesized S-galactosyl oligo(Arg) conjugates to overcome the serious problem of the passage through the E. coli cell membrane. Following in vivo expression of green fluorescent protein (GFP) induced by each of the S-galactosyl (Arg)n constructs (n = 5, 6, 8) at the T5 promoter in E. coli for 18 h, we visually observed that the cultures fluoresced green light when excited with UV light. The fluorescence intensities for these cultures were greater than that found for a control culture, indicating that the peptides had induced GFP expression. In order to accomplish higher expression efficiency, we investigated the cluster effect and structural fine-tuning of new poly(2-oxazoline) containing CysArgArg as the cell-penetrating peptide (CPP) and S-galactosides when acting as inducers of recombinant protein expression under the control of lac operator/repressor systems in this article. Quantitative fluorescence intensities (calculated per molecule) also supported the observations that the cell-penetrating glyco poly(2-oxazoline)s were better inducers of GFP expression than glyco poly(2-oxazoline) containing no CPP or isopropyl β-d-thiogalactoside. Because the level of GFP expression was directly related to the number of sugar residues in each glyco poly(2-oxazoline), we propose that a cluster effect of the S-galactosides attached to the cell-penetrating poly(2-oxazoline) is responsible for how well the galactosides inhibited the lac repressor to activate the protein expression under the control of the lac operator/repressor system. A similar tendency was observed when the T7 promoter was placed upstream of the gene for an artificial extracellular matrix protein and glyco poly(2-oxazoline)s-CPP conjugates were used as inducers. To assess how the glyco poly(2-oxazoline) penetrate the cell membrane, we labeled the glyco poly(2-oxazoline) using 1-amino pyrene and directly observed the penetration process. Furthermore, we could visualize protein expression under the control of a lac promoter/operator/repressor system using transmission electron microscopy combined with energy dispersive X-ray analysis mapping.

Cation−π Interactions Accelerate the Living Cationic Ring-Opening Polymerization of Unsaturated 2-Alkyl-2-oxazolines

“…These functionalities are of interest for various click reactions, such as copper­(I)-catalyzed azide cycloaddition as well as thiol–ene and thiol–yne reactions, which enable facile postfunctionalization of the resulting polymers. Click reactions on PAOx are typically performed by using copolymers and have found many practical applications, such as the introduction of targeting molecules on polymeric chains, coatings of nanoparticles − or stainless steel, − creation of biocompatible materials with anode-selective deposition behavior, , and many others. ,− In addition, postpolymerization modification readily enables the introduction of side-chains that are not compatible with the CROP of 2-oxazolines . Moreover, cross-linking of alkene- and/or alkyne-functionalized PAOx has been applied for the development of hydrogels or core–shell cross-linked nanoparticles. ,,− …”

Glycopolymer Grafted Silica Gel as Chromatographic Packing Materials

“…The synthesis of sugar-based monomers and polymers has been widely reported over the last two decades. The carbohydrate residues confer high hydrophilicity and water solubility and they are widely applied in macromolecular drugs [1], drug delivery [2], cell targeting, and adhesion [3,4]. Use of such polymers as stationary in separation applications and bioassays has also been suggested for structure-controllable sugar polymers [5].…”