Synthesis of Glycopolymer Containing Cell-Penetrating Peptides as

Apr 8, 2016 - Furthermore, we could visualize protein expression under the control of a lac promoter/operator/repressor system using transmission elec...
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Synthesis of Glycopolymer Containing Cell-Penetrating Peptide as Inducers of Recombinant Protein Expression under the Control of Lac Operator/Repressor Systems Kei Katagiri, Akinori Takasu, and Masahiro Higuchi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00368 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 10, 2016

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(Article) Synthesis of Glycopolymer Containing Cell-Penetrating Peptide as Inducers of Recombinant Protein Expression under the Control of Lac Operator/Repressor Systems

∗[a]

Kei Katagiri, Akinori Takasu

, Masahiro Higuchi

Department of Life Science and Applied Chemistry Nagoya Institute of Technology Gokiso-Cho, Showa-Ku, Nagoya 466-8555, Japan Fax: (+) 81-52-735-5266 E-mail: [email protected]

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 hours, 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 (IPTG). 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 RNA transcription process. Furthermore, we could visualize protein expression under the control of a lac promoter/operator/repressor system using transmission electron microscope (TEM) combined with energy dispersive X-Ray analysis (EDX) mapping.

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Introduction Cell-penetrating peptides (CPPs), highly cationic peptides usually rich in arginine and lysine amino acids, are characterized by their ability to translocate quickly into almost any live cell.1,2 Some sequences, such as the protein transduction domain from HIV transactivator of transcription (TAT) protein (GRKKRRQRRRPPQ),3 can be recognized by the nuclear pore complexes (NPCs)4 and thus can actively transport proteins,5 DNA,6,7 nanoparticles,2,8 and other cargos from the cytosol into cell nuclei. Therefore, CPPs have been used as exceptionally efficient “locomotives” for intracellular and nuclear delivery of various cargos ranging from small molecules such as anticancer drugs9 to macromolecules, for instance, full-length proteins and peptides10 and even nanoparticles.11,12 The RNA polymerase of bacteriophage (T5/T7) has a strict specificity for its own promoters. In 1961, Jacob and Monod suggested the use of the Escherichia coli lactose (lac) operon as a model for gene regulation.13 This model system is still used to study how a structural gene set can be coordinately transcribed or repressed, depending upon the metabolites found in the intercellular environment. The lac repressor contains 360 amino acids and associates to form a homotetramer of 154,520 Da.14 Each monomer contains one saccharide binding site (Figure 1) and from the reported X-ray diffraction analysis of lac repressor,15,16 we estimated the distance between the sugar-binding sites to be 2.4 nm. Notably, isopropyl β-D-thiogalactoside (IPTG)—which is not a substrate for β-galactosidase, but a molecular mimic of allolactose derived from lactose, which may be the “true” inducer—acts as a gratuitous inducer and turns on transcription of the lactose operon via its interaction with the lac repressor.13 IPTG permeates E. coli without the assistance of the lac permease.17

Figure 1. Structures of lac repressor and polymeric inducer-lac repressor complex.

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Much attention is currently being devoted to glycobiology and glycochemistry topics in biomedicine and biochemistry,18–26 especially as they relate to interactions between proteins and carbohydrates. Carbohydrate-protein18–24 and carbohydrate-carbohydrate25,26 interactions have been well characterized, and notably, such interactions are often strengthened by the presence of multiple binding sites. Certain glycopolymers in which multiple saccharide residues are incorporated into their polymer backbones have enhanced binding affinities toward their targeted proteins in comparison with their monomeric, saccharide-containing building blocks. This property has been ascribed to multivalent recognition, i.e., the cluster effect.18–24 Notably, certain glycopolymers, in which the saccharide spacing is random, strongly bind their target proteins.18–24 Aoi et al.27,28 used a dendrimer skeleton and Matsuura et al.29,30 reported alternative strategies to prepare periodic glycosylated oligonucleotides (20-mers) as a means of controlling the three-dimensional arrangements of the pendant saccharides. These carbohydrate-containing compounds bound strongly to certain lectins. We have also described the binding affinities of α-helical peptides that contained a pendant saccharide linked to the peptide backbone via an O-glycoside linkage31 (a model for mucine-type glycoproteins and glycopeptides) or via an N-glycoside linkage.32,33 Recent progress involving glyco-conjugates for biomedical applications, e.g., drug delivery systems, anti-bacterial activity, inhibition of viral infection, and the spread of malignant tumors and the human immunodeficiency virus has been remarkable with many excellent results.18 However, as far as we know, there have been only a few studies concerning the design of glyco-conjugates that can act as inducers of recombinant protein expression under the control of a lac promoter/operator/repressor system.34,35 Furthermore, as far as we know, there have been no reports focused on the cluster effect of saccharides or allosteric effects of the repressor proteins until now. Recently we synthesized S-galactosyl dendrimers34 and S-galactosyl poly(oxazoline)s prepared by ring-opening polymerization of 2-oxazoline containing a S-galactoside as the substituent,35 which we expected to act as inducers of recombinant protein synthesis via the cluster effect. However, their limited ability to permeate the E. coli membrane precludes their use as inducers. We also designed S-galactosyl-oligo(arginine) [S-Gal-oligo(Arg)] conjugates, including one containing the “magic arginine number” eight (R8),36-38 to use as new types of lac operon inducers.39 In order to accomplish higher expression efficiency, we investigated the cluster effect and structural fine tuning of glyco poly(2-oxazoline)s-CPP conjugate (Figure 2) as inducers of recombinant protein expression under the control of lac operator/repressor systems in this article.

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Figure 2. Chemical Structure for Glyco Poly(2-oxazoline)s-CPP Conjugate.

Experimental Section Materials. 1-Thio-β-D-galactose tetraacetate was purchased from Sigma-Aldrich Corp. Fluorenylmethyloxycarbonyl chloride (Fmoc) / 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf)-protected Arg-CLEAR-Acid-Resin, Fmoc-Arg(Pbf)-OH, and Fmoc-Cys(Trt)-OH, were purchased from the Peptide Institute, Inc. 1-Hydroxybenzotriazole (HOAt) and N,N'-diisopropylcarbodiimide (DIPCI) were purchased from Watanabe Chemical Industries. Piperidine, trifluoroacetic acid, hydrazine monohydrate, and all other reagents were analytic grade and purchased from Japanese companies; these compounds were used without further purification. Measurements. 1H- and 13C-NMR spectra were recorded at 27 °C using a Bruker DPX200 spectrometer (400 MHz for 1H and 100 MHz for 13C). Chemical shifts were referenced to the tetramethylsilane signal (δ = 0). The number-average molecular weight (Mn) and the polydispersity index (Mw/Mn) of each polymer were determined by size exclusion chromatography (SEC) using a Tosoh DP8020 pump system, a refractive index (RI) detector (Tosoh RI-8020), and either a TSKgel SuperMultiporeHZ-M column (eluent, chloroform; flow rate, 0.35 mL/min; temperature, 40 °C; Tosoh Corp.), in which poly(styrene)s are used as the calibration standard. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were recorded using a JMS-S3000 mass spectrometer (JEOL), with α-Cyano-4-hydroxycinnamic acid as the matrix agent. NaI was used to generate monosodium-cationized ions of the CysArg2 derivatives ([M+Na]+). Fluorescence spectrometry were measured using Fluorescence Spectrophotometer F-2700 (HITACHI). Fluorescence microscopy was performed using OLYMPUS BX51 equipped with fluorescence mirror unit (U-MWU2). Transmission electron microscope (TEM) combined with energy dispersive X-Ray analysis (EDX) mapping was performed using JEM-z2500 (JEOL). Preparation of 2-(3-Butenyl)-2-oxazoline. 2-(3-Butenyl)-2-oxazoline was synthesized via N-(2-chloroethyl)-4-pentenamide according to the reported method40-42 and the final product was distilled to give 2-(3-butenyl)-2-oxazoline as a colorless liquid in 34% yield (2.42 g). 1 H NMR (200 MHz, CDCl3, δ): 2.37 (4H, CH2=CH-CH2CH2-), 3.82 (t, 2H, J = 9.3 Hz, =N-CH2CH2-), 4.22 (t, 2H, J = 9.3 Hz, -O-CH2CH2-), 4.95−5.14 (2H, CH2=CH-), 5.73−5.97 (1H, CH2=CH-). Cationic Ring-Opening Polymerization of 2-(3-Butenyl)-2-Oxazoline. Methyl triflate was stirred in a dry, 10 mL round-bottom flask with acetonitrile and 2-(3-butenyl)-2-oxazoline [7.95 mmol, initial monomer concentration ([M]0): 1 M; [M]0/[I]0= 20/1, initial initiator concentration ([I]0)] at 70 °C for 48h under nitrogen. The reaction mixture was then allowed to cool to room temperature, and 1M NaOH/Methanol (MeOH) (1.19 mmol, 3 equiv relating to the initiator) was added to terminate the reaction. The mixture was stirred at room temperature overnight, polymer was purified by precipitation into excess amount of ice-cold diethyl ether. The yield of polymer was 66.1 % yield (0.658 g, Mn=1,719 , Mw/Mn= 1.54) 4 ACS Paragon Plus Environment

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H NMR (400 MHz, CDCl3, δ): Poly[2-(3-butenyl)-2-oxazoline]: 2.15−2.56 (4H, br, -C=OCH2CH2-), 3.19−3.64 (4H, br, -NCH2CH2N-), 4.89−5.16 (2H, br, -CH=CH2), 5.65−5.98 (br,-CH=CH2). Preparation of Cys(Arg)2. Protected Cys(Arg)2 were first prepared by solid-phase synthesis using an EYELA Solid Organic Synthesizer CCS-150M. Amino acids were coupled via activation with HOAt, and DICPI. Fmoc deprotection was performed using 20% piperidine in N,N-dimethylformamide (DMF). Removal of Fmoc-Cys(Trt)-[Arg(Pbf)]2-resin preparations from the resin and deprotection of the arginine and cysteine side chains were performed simultaneously by adding ice-cold trifluoroacetic acid (19 mL), H2O (1.0 mL), 1,2-ethanedithiol (1.7 mL), and thioanisole (1.0 mL) to each ice-cold peptide solution and then stirring each mixture at room temperature for 3 hours. The mixture was then filtered, and each filtrate was subjected to reduced pressure to remove any remaining trifluoroacetic acid. The filtrate was poured into 200 mL of ice-cold diethyl ether. The precipitate was lyophilized to afford white powder (91% yield). 1 H NMR (400 MHz, D2O, δ): Cys(Arg)2]: 1.59−1.95 (8H, -CH2CH2-), 3.01−3.17 (2H, HS-CH2CH-), 3.17−3.30 (4H, NH=C-NH-CH2-), 4.24−4.42 (2H,-C=OCHNH-). Thiol-ene Functionalization with Cys(Arg)2. Poly[2-(3-butenyl)-2-oxazoline] (25 mg, 0.20 mol 1 equiv) was dissolved in 0.67 mL of DMF. Cys(Arg)2 (21.68 mg, 0.05 mmol, 0.25equiv) and 1-hydroxycyclohexyl phenyl ketone (Irgacure 184; 5.11 mg, 0.025 mmol 0.05equiv) were added, and the mixture was degassed and replaced with nitrogen and exposed to UV light using a Toshiba H-400P high-pressure mercury lamp (400 W and 250−400nm) for 8 h. The solvent was concentrated in vacuo. The residue was dissolved in H2O, filtered and poly [2-(3-butenyl)-2-oxazoline] containing 25% Cys(Arg)2 was obtained upon lyophilization. 1 H NMR (400 MHz, DMSO, δ): Poly[2-(3-butenyl)-2-oxazoline containing Cys(Arg)2]: 1.45−1.75 (br, CH2 in side chains), 2.15−2.35 (br, CH2 in side chains), 2.65−3.00 (br, -CH2 -S- ), 3.20−3.45 (4H, br, CH2 in backbone), 4.10−4.40 (1H, br, NH-CH-C=O-), 4.85−5.16 (br, CH2=CH- side chains), 5.71−5.96 (br, CH2=CH- side chains). Thiol-ene Functionalization of Poly(2-oxazoline) Having Pendent CPP with 1-Thio-β β-D-Galactose Tetraacetate. Poly[2-(3-butenyl)-2-oxazoline] containing 25% of pendent Cys(Arg)2 was dissolved in DMF. 1-Thio-β-D-galactose tetraacetate (109.3 mg, 0.3 mmol, 1.5 equiv) and 1-hydroxycyclohexyl phenyl ketone (Irgacure 184; 15.3 mg, 0.075 mmol, 0.3 equiv) were added, the mixture was degassed and replaced with nitrogen and exposed to UV light using a Toshiba H-400P high-pressure mercury lamp (400 W and 250−400nm) for 8 h. The solvent was concentrated in vacuo. The polymer was dissolved in MeOH and was purified by precipitation into an excess amount of H2O (81.2 % yield Mn=10,490 , Mw/Mn= 1.18). The observed Mn=10,490 did not show any discrepancies with the calculated Mn of 6,981. 1 H NMR (400 MHz, DMSO, δ): glyco(Ac)4 poly[2-(3-butenyl)-2-oxazoline containing Cys(Arg)2]: 1.32−1.75 (br, CH2 side chains), 1.87−2.12 (m, acetyl group), 2.25−2.42 (br, CH2 side chains), 3.31−3.60 (4H, br, CH2 backbone), 3.92−4.33 (m, sugar H-5 and 6), 4.80-5.17 (m, sugar H-1, 2, and 3), 5.20−5.43 (m, sugar H-4). To deprotect the acetyl groups of glyco poly(oxazoline)s containing cell-penetrating peptides, the product was dissolved in DMF containing hydrazine monohydrate ([hydrazine]0/[acetyl group]0=5/1) and stirred at room temperature for 8 h. After the reaction, acetone was added to the solution, and the mixture 5 ACS Paragon Plus Environment

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was stirred for 2 h to quench the reaction. The reaction mixture was evaporated in vacuo, the residue was dissolved in H2O and was purified by precipitation into an excess amount of MeOH. After dialysis for 3days (MWCO = 1000), a white powder was obtained using freeze-dryer (17.5 % yield). 1 H NMR (400 MHz, D2O, δ): glyco poly[2-(3-butenyl)-2-oxazoline containing pendent Cys(Arg)2]: 1.22−1.75 (br, CH2 in side chains), 2.15−2.42 (br, CH2 in side chains), 2.55−2.76 (br, -CH2 -S- side chains), 3.24−3.50 (4H, br, CH2 in backbone), 3.05−3.64 (m, sugar H-2,3.5 and 6 ), 3.72-3.85 (m, sugar H-4), 4.15−4.26 (br, NH-CH-C=O-), 4.35−4.46 (m, sugar H-1). Expression of Green Fluorescent Protein (GFP) in E. coli via a T5 Expression System under the control of a lac promoter/operator/repressor system. E. coli K10 cells were transformed with a pQE9-GFP2/pREP4 vector containing the gene for GFP for which expression was under the control of a lac promoter/operator/repressor system.34,35 The cells were cultured at 37 °C in M9 medium (5 mL) supplemented with 0.2% (w/v) glucose, 35 mg/mL thiamine, 0.1 mM MgSO4, 0.1 mM CaCl2, the 20 common amino acids (4 mg/mL each), 25 mg/mL kanamycin, and 100 mg/mL ampicillin to an OD600 nm of 0.6, after which the culture was divided into aliquots for the expression experiments. GFP expression was assessed at 37 °C by adding glyco poly(2-oxazoline)s-CPP conjugate or IPTG into a culture (each at final concentration, 1 mM) and incubating the cultures for 4h and 8h, after which the fluorescence of each was visualized by UV light irradiation (395 nm). A culture to which no peptide or inducer had been added served as the negative control. To quantify expression levels, the fluorescence of each culture was measured at 509 nm (exctation at 395 nm) using a Hitachi F-2700 spectrometer. The fluorescence (F) of each culture, expressed as ∆ (F/OD), was normalized to the number of cells (F/OD600 43,44 The F/OD were calculated per molecule (standard: IPTG=1000). ∆ (F/OD) = [F ]/OD nm). 600 509 600 (after induction) – [F ]/OD s (before induction). At least three independent experiments were carried 509 600 out in order to check the reproducibility. Expression of artificial extracellular matrix protein aECM-CS5-ELF-F in E. coli via a T7 Expression System under the control of a lac promoter/operator/repressor system. The pET28-CS5-ELF-PheRS* plasmid in which a linker sequence encoding a T7-tag, a hexahistidine tag, and an enterokinase cleavage site was cloned into a pET28 plasmid between its Nco I and Xho I sites, was transformed into the phenylalanine-auxotrophic E. coli BL21(DE3) strain AF [HsdS gal (ncIts857 ind 1 Sam7 nin5 lacUV5-T7 gene 1) pheA], which was constructed in the Tirrell laboratory. This E. coli strain AF-IQ[pET28-CS5-ELF-PheRS*] served as the expression system. The cells were cultured at 37 °C in M9 medium (5 mL) supplemented with 0.2% (w/v) glucose, 35 mg/mL thiamine, 0.1 mM MgSO4, 0.1 mM CaCl2, the 20 common amino acids (4 mg/mL each), 25 mg/mL kanamycin, and 20 mg/L chloramphenicol to an OD600 nm of 0.6, after which the culture was divided into aliquots for the expression experiments. The expression was assessed at 37 °C by adding glyco poly(oxazoline)s containing cell-penetrating peptides or IPTG into a culture (each at final concentration, 1 mM) and incubating the cultures for 4h. Protein expression was monitored by SDS polyacrylamide gel electrophoresis (PAGE) using a normalized OD600 of 0.5 per sample and TEM-EDX mapping.

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Results and Discussion We synthesized, using a solid-phase strategy, Cys(Arg)2 (Scheme 1) as the cell-penetrating trigger. The yield of the peptide was 91%, and the structure was confirmed by 1H NMR and MOLDI-TOF mass spectra (see Figure S1,S2). After ring-opening polymerization of 2-(3-butenyl)-2-oxazoline, the peptide [Cys(Arg)2] was coupled to the poly(2-oxazoline) backbone via thiol-ene click chemistry, followed by another thiol-ene addition using 1.5 eq. of 1-thio-β-D-galactose tetraacetate in order to consume the remaining double bonds in the backbone (Scheme 2). 1H-NMR spectroscopy (400 MHz, Supporting Information, Figure S3) agreed with the expected structure in all cases. The deacetylated products were completely water soluble. Scheme 1. Preparation of Cys(Arg)2.

Scheme 2. Synthesis of Glyco Poly(2-oxazoline)s-CPP Conjugate. O OH

N

N

H3 C

n O

MeOTf 60°C

N

NaOH/MeOH

O

OH

H3C

N

CysArgArg

n

S NH2

8h, 365nm, r.t.

O

O

HN

H N

O

NH2

NH 2 NH

NH N H

HN

HO

OH

O

OH

OAc OAc HO

AcO O OAc

OAc O

O SH

AcO OAc

O

OAc O

S OH

N

Hydrazine/DMF

N

H3C

n

S OH

N

N

H 3C

n

8h, r.t.

O

8h, 365nm, r.t.

O

S

S NH 2

HN O NH 2 NH HN

N H

O

NH 2

H N

HN NH 2

O

O

O

H N

NH 2

NH 2

NH

NH HN

HO

OH

NH

N H HO

O

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Fluorescence assays based on green fluorescent protein (GFP) in E. coli cells containing pQE9-GFP2/pREP4 were performed to evaluate the abilities of the glyco poly(2-oxazoline)s containing cell penetrating peptides to act as inducers of GFP expression. The cells were cultured at 37 °C in M9 medium (5 mL). For each assay, when the OD600 nm of a culture (5 mL) was 0.6, one of either of the cell-penetrating glycopolymers or IPTG (each at 1 mM) was added. After an 4-8 h induction, the visible fluorescence of each culture was observed (irradiation at 365 nm, Figure 3), quantified by fluorescence spectrometry (excitation at 395 nm and emission at 509 nm), and normalized to the OD600 nm of each culture (F/OD600 nm; Figure 4).27,28 As expected, the normalized fluorescence of the culture was relatively low when the glyco poly(2-oxazoline) without CPP was the inducer (Figure 5, and see also Figures S4) . Using the S-glyco poly(2-oxazoline) ([sugar]/[CPP]=75/25, Mn= 4490) peptides as inducers, fluorescence at 509 nm was detected (F/OD600 = 1649), which is larger even than that for IPTG (F/OD600 = 1000) as shown in Figure 5. The results indicated that a sugar-cluster effect18–24 was also in operation for recombinant protein under the control of the lac repressor/operator system, of which this is the first reporting. The fluorescent measurements also showed that the glyco poly(2-oxazoline)s ([sugar] /[CPP]=75/25, and 46/54: Mn= 5350) were better inducers than were the galactosyl dendrimers (F/OD600 = 145)34 and the poly(oxazoline)s35 (F/OD600 = 588) after 4h incubation. Because GFP expression increased in the presence of oligo(Arg) peptides,39 it seems that an introduction of arginines improved the passage of the glycopolymers through the E. coli cell membrane. On the other hand, since oligoarginine peptides are well-known to enhance gene transfection efficiency in various cell lines,45-47 we now have to eliminate the possibility that oligoarginine peptides can enhance protein expression in E. coli cells. The cell penetrating poly(2-oxazoline) containing CPP but no S-galactoside (Mn= 6,703) and CPP (Figure S5) were prepared and used as the inducers for both of the expression of GFP under the same conditions. After 4 h expression, the green fluorescences were at a trace level in the fluorescent measurements (Figure S5) and it can be concluded that there is a possible synergistic effect between the galactosyl moiety and the oligoarginine peptides as we expected.

Figure 3. Pictures of M9 culture after 4 h induction (UV irradiation).

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Figure 4. Normalized fluorescent intensities (F/OD600 nm) of E. coli (T5 promoter) cultures after 4h induction with IPTG and several inducers which had different ratios (CPP:sugar). The F/OD600s were calculated per molecule (standard: IPTG=1000). ∆ (F/OD) = [F509]/OD600 (after induction) – [F509]/OD600 (before induction).

Figure 5. Normalized fluorescent intensities (F/OD600 nm) of E. coli (T5 promoter) cultures after 4h induction with IPTG and several inducers which had different ratios (CPP:sugar). The F/OD600s were calculated per molecule (standard: IPTG=1000). ∆ (F/OD) = [F509]/OD600 (after induction) – [F509]/OD600 (before induction). 9 ACS Paragon Plus Environment

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Next, we used a pET expression system that included the gene for an artificial extracellular matrix (ECM) protein (aECM-CS5-ELF-F) that contains a fibronectin cell-binding domain (CS5) and an elastin sequence (ELF).39 To assess the ability of the glyco poly(2-oxazoline) to act as inducers aECM-CS5-ELF-F was expressed in E. coli using a T7 expression system.39 The T7 promoter and its downstream target gene, reside in a pET plasmid. Residual T7 polymerase activity is inhibited by T7 lysozyme, which is constitutively expressed at low levels, and the gene for which is present in either the pLysS or pLysE plasmid.39 The pET system can produce large amounts of ECM protein (up to 100 mg/L of culture medium). For this study, the glyco poly(2-oxazoline) ([sugar]/[CPP]=75/25) was used as the inducer of aECM-CS5-ELF-F expression,39 which was controlled by a T7 promoter upstream of a lac operator. Whole cell lysates and purified aECM-CS5-ELF-F were subjected to SDS polyacrylamide gel electrophoresis (PAGE). Substantial amounts of a protein with a calculated molecular weight of 42,600 that corresponds to that of aECM-CS5-ELF-F were seen (Figure S6), indicating that the lac operator was activated by the glyco poly(2-oxazoline) in the T7 expression system. Finally, to investigate whether the glyco poly(2-oxazoline) penetrates into the cell membrane, we labeled the glyco poly(2-oxazoline) ([sugar]/[CPP]=46/54) using 1-amino pyrene and directly observed the cell-penetration process using fluorescence spectroscopy. In Figure 6, cells were observed after induction by the pyrene-labeled poly(2-oxazoline) using a fluorescence microscope (at 420 nm) (Figure 6A) as well as a normal microscope (Figure 6B). A strong blue light characteristic of the linked 1-amino pyrene was observed to be emitted from within the cells, suggesting that the pyrene-labeled glyco poly(2-oxazoline) had accumulated in the cells (Figure 6A). Substantial amounts of a protein with a calculated molecular weight of 42,600, corresponding to that of aECM-CS5-ELF-F, were detected by SDS-PAGE, indicating that the lac operator was activated by the labeled glyco poly(2-oxazoline)s-CPP conjugate in the T7 expression system, and no localized concentrations of blue fluorescence were observed in the presence of non-labeled glyco poly(2-oxazoline)s-CPP conjugate (data not shown). From the results, it seems that the new CPP-containing inducers actually went into the E. coli cells.

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A

B

Figure 6. Fluorescence microscope (Figure 6A) and normal microscope (Figure 6B) images of E. coli after induction by pyrene-labeled glyco poly(2-oxazoline) containing pendant CPP (CPP:sugar=46:54). Furthermore, we can visualize protein expression under the control of a lac promoter/operator/repressor system using TEM-EDX mapping (Figure 7-9). For each assay, when the OD600 nm of a culture (5 mL) was 0.6, one of either of IPTG or the cell-penetrating glycopolymer([sugar]/[CPP]=75/25) (each at 1 mM) was added. After an 4 h induction, we observed cells on the Elastic Carbon Grid ELS-C10. The colors in the TEM-EDX mapping identify carbon (red), nitrogen (green), sulfer (blue), phosphorus (purple) and oxygen (yellow). Carbon and nitrogen are from protein, and phosphorus and oxygen are form RNA. Without induction as the negative control (Figure 7), all of elemental compositions were uniform. On the other hand, in Figures 8 and 9, we observed local high concentration of carbon (red) and nitrogen (green) after induction by IPTG (positive control) and glyco poly(2-oxazoline)s-CPP conjugate, respectively. The results indicated that the encoded protein (aECM-CS5-ELF-F) are biosynthesized in the cells. 11 ACS Paragon Plus Environment

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7.. The TEM-EDX mapping images of E.coli with no induction after 4h (negative control). Figure 7

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Figure 8. The TEM-EDX images of E.coli with induction using IPTG after 4h (positive control).

Figure 9. The TEM-EDX images of E.coli with glyco poly(2-oxazoline)s-CPP conjugate after 4h. 13 ACS Paragon Plus Environment

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Conclusion In our laboratory, we had previously synthesized glyco poly(oxazoline)s as inducers of recombinant protein expression. Using Escherichia coli with a T5 promoter for expression of green fluorescent protein, we found they induced expression of only small amounts of GFP, because of their inability to efficiently penetrate the Escherichia coli cell membrane. To overcome this problem, we synthesized glyco poly(2-oxazoline)s containing cell-penetrating peptides. We examined the transcription activity under the control of a lac promoter/operator/repressor system. Quantitative fluorescent measurements also supported the observations that the glyco poly(2-oxazoline)s-CPP conjugates were better inducers of GFP expression than glyco poly(2-oxazoline) or isopropyl β-D-thiogalactoside with respect to the expression level per molecule. As the level of GFP expression was directly related to the number of sugar residues in each cell-penetrating poly(2-oxazoline) (containing between two and four galactosides), we propose that a cluster effect of the S-galactosides attached to glyco poly(2-oxazoline) with cell-penetrating peptide 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 the glyco poly(2-oxazoline)s-CPP conjugates were used as inducers. To assess how the glyco poly(2-oxazoline)s penetrate the cell membrane, we labeled the glyco poly(2-oxazoline) using 1-amino pyrene and could directly observed the RNA transcription process.

Acknowledgements We are grateful to Prof. David A. Tirrell (California Institute of Technology) and Dr. Inchan Kwon (Gwangju Institute of Science and Technology) for the gift of the E. coli K10 GFP expression system, as well as helpful suggestions and discussion. This work was funded by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Development Scientific Research, No. 15K05515) and Japan Science and Technology (JST) “Research for Promoting Technological Seeds”. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The data for the NMR and MALDI-TOF mass spectra of CPP and glycopolymers, and SDS-PAGE of aECM-CS5-ELF-F from cell lysate (PDF).

Keywords: (glyco poly(2-oxazoline), cell-penetrating peptide, lac operon, biosynthesis, repressor)

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Entry for the Table of Contents

FULL PAPERS Kei Katagiri, Akinori Takasu

Synthesis of Glycopolymer Containing Cell-Penetrating Peptide as Inducers of Recombinant Protein Expression under the

Control

of

Lac

Operator/Repressor Systems

IPTG

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