Chemical Cleavage of an Asp-Cys Sequence Allows Efficient

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Chemical cleavage of Asp-Cys sequence allows efficient production of recombinant peptides with an N-terminal cysteine residue Katia Pane, Mariavittoria Verrillo, Angela Avitabile, Elio Pizzo, Mario Varcamonti, Anna Zanfardino, Antimo Di Maro, Camilla Rega, Angela Amoresano, Viviana Izzo, Alberto Di Donato, Valeria Cafaro, and Eugenio NOTOMISTA Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00083 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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Bioconjugate Chemistry

Chemical cleavage of Asp-Cys sequence allows efficient production of recombinant peptides with an N-terminal cysteine residue

Authors: Katia Pane§¶, Mariavittoria Verrillo†¶, Angela Avitabile§, Elio Pizzo§, Mario

Varcamonti§, Anna Zanfardino§, Antimo Di Maro‡, Camilla Rega‡, Angela Amoresano#, Viviana Izzo∞, Alberto Di Donato§, Valeria Cafaro§&, Eugenio Notomista§&*

§

Department of Biology, Università degli Studi di Napoli Federico II, Via Cintia 4, Napoli,

80126, Italy †

Department of Agricultural Sciences, Università degli Studi di Napoli Federico II, Via

Università 100, Portici, 80055, Italy ‡

Department of Environmental, Biological and Pharmaceutical Sciences and Technologies,

Università della Campania Luigi Vanvitelli, Via Vivaldi 43, Caserta, 81100, Italy #

Department of Chemical Sciences, Università degli Studi di Napoli Federico II, Via Cintia 4,

Napoli, 80126, Italy ∞

Department of Medicine and Surgery, Università degli Studi di Salerno, via S. Allende,

Baronissi, 84081, Italy

¶

These authors contributed equally to this work.

&

These authors also contributed equally to this work.

*Corresponding author: Dr. Eugenio Notomista, Department of Biology, University of Naples Federico II, Via Cintia 4, 80126, Naples, Italy (email: [email protected]).

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Abstract Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoBL,, an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as function of pH, temperature and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60°C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%) and the expected content of free thiol groups (1 mole per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue. 2


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Keywords: protein chemical cleavage, N-terminal cysteine, recombinant peptide production, antimicrobial peptide, thiol labeling, amino thiols.

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Introduction Peptides carrying an N-terminal cysteine residue are frequently used in several medical, analytical and industrial applications, due to a very unique reactivity that allows site-specific N-terminal modification. Terminal cysteine residues have been successfully used to obtain directional immobilization thus preserving biological properties of biomolecules1,2, to label and/or functionalize peptides3,4 and to perform the chemical synthesis of proteins.5,6 Figure 1 compares some of the most popular strategies for cysteine modification (panel A) like formation of mixed disulfides (reaction 1) and alkylation by maleimide and haloacetic acids derivatives (reactions 2 and 3, respectively), and reactions typical of Nterminal cysteine residues (panel B) like “natural chemical ligation” (NCL) and cyclization mediated by 2-cyanobenzothiazole (CBT) derivatives (reactions 4 and 6, respectively) which can be selectively performed also in the presence of internal cysteine residues. In particular, 2-cyanobenzothiazole7,8 has been widely used to selectively conjugate fluorophores3,9, radioactive4,10, magnetic tags2 or microarrays1 to the N-terminus of proteins and peptides11, developing new techniques for imaging, drug discovery and disease diagnosis. When using peptides for analytical, diagnostic and industrial purposes, one of the major methodological limits is to obtain the peptides with cost-effective procedures. Peptides with an N-terminal cysteine residue can be produced by standard solid-phase peptide synthesis.12 However, costs are generally high and when the peptides are longer than 30 amino acids yields are low.

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Figure 1. Examples of reactions for cysteine modification. Panel A shows three popular reactions for the modification of terminal or internal cysteine residues: formation of a mixed disulfides (1) alkylation by substituted maleimides (2), alkylation by substituted haloacetamides (3). Panel B shows three reactions typical of N-terminal cysteine residues: reaction with thioesters (4), cyclization with aldehydes (5), cyclization with 2cyanobenzothiazoles (6). P1 can be a hydrogen atom or the polypeptide chains upstream to the cysteine residue. P2 can be a OH group or the polypeptide chain downstream to the cysteine residue. R1 and R2 can be a wide variety of groups including labels, fluorophores, protein/peptides and solid supports. R3 usually is a small alkyl or aryl group. EW is an electron withdrawing group (e.g. the nitrobenzoate group in the Ellman reagent). X is iodide or bromine. Cysteine bonds and atoms are shown in bold for clarity.

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To overcome limitations of solid-phase synthesis, recombinant techniques have been developed. One of the most widely used method is based on the production of peptides as fusion proteins in E. coli.13-17 Release of the desired peptide with an N-terminal cysteine residue is usually obtained by enzymatic18 or chemical cleavage.14 For example, thrombin can cleave a modified consensus sequence (LVPR-C)19, although with less efficiency with respect to the canonical site (LVPR-G). Also Factor Xa and enterokinase can be used to generate peptides with an N-terminal cysteine residue because they mainly interact with residues located at the N-terminus of the hydrolyzed bond. However proteases, do not always yield high cleavage efficiency and, furthermore, they can cleave fusion proteins also at noncanonical sites.18,19 Another enzymatic approach to the release of the desired peptide from the fusion protein takes advantage of E. coli endogenous methionine aminopeptidases20 to cleave the initiating methionine from a recombinant protein containing cysteine as the second residue. However, removal of methionine has been found to be incomplete and related to the nature of the following amino acids.20 Furthermore, the recombinant proteins are sometimes obtained with undesired modifications due to reaction of the N-terminal cysteine with cell metabolites.20 Moreover, it should be taken into account that all these enzymatic methods require a soluble fusion protein and that the cleavage site is accessible to the enzyme. This makes enzymatic cleavage methods not suited especially when the peptides are toxic for the E. coli host cells, or very hydrophobic or scarcely soluble. In these cases, the most efficient strategy is to fuse the peptide to a carrier which drives it into inclusion bodies.14 This not only protects host cells but also reduces the risk of proteolytic degradation of the peptide. When peptides 6


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are produced as insoluble fusion proteins, release of the peptide is generally obtained by nonenzymatic chemical cleavage methods under denaturing conditions.14 Among commonly used chemical cleavage methods only cyanogen bromide21,22 allows preparing peptides with an Nterminal cysteine. This is possible by inserting a Met-Cys sequence between the carrier and the peptide. Besides the toxicity, other disadvantage of cyanogen bromide cleavage are that the peptide/protein of interest should not contain internal methionine residues and the fact that usually this method requires harsh conditions which can determine undesired modifications of side chains.14 Recently, we have described a very efficient method to produce cationic antimicrobial peptides (CAMPs) in E. coli as insoluble fusion proteins23 with a carrier, named ONCDCless-H6, derived from the M23L mutant of onconase (ONC), a frog RNase.24,25 Efficient release of peptides (95%) is obtained by acid hydrolysis of an Asp-Pro sequence23 inserted between the carrier and the desired peptide under very mild conditions (0.1 M acetic acid, pH 2.0 at 60°C). This method has been successfully used to prepare an antimicrobial peptide located at the C-terminus of human thrombin, named GKY2023,26,27, an antimicrobial peptide corresponding to residues 887-923 of human Apolipoprotein B, named ApoBL28, and an antimicrobial peptide corresponding to residues 133-150 of human Apolipoprotein E29, with yields of 10-20 mg/L of culture and a purity of about 95% or higher. Unexpectedly, during the development of the carrier we found that two Asp-Cys sequences present in (M23L)ONC underwent acid mediated cleavage with an efficiency comparable to that of Asp-Pro sequences23, whereas other Asp-X sequences present in the protein showed very limited hydrolysis. Although acid Asp-X cleavage of peptides and proteins has been extensively studied30-32, to the best of our knowledge the higher sensitivity to hydrolysis of the Asp-Cys with respect to other Asp-X sequences has never been reported before.

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Given the interest of this cleavage for the production of peptides with a cysteine at the N-terminus we wondered if acid labile Asp-Cys sequence could be used to obtain the release of peptides containing an N-terminal cysteine from a carrier protein. To this purpose, we produced two fusion proteins, ONC-DCless-H6-(C)GKY20 and ONC-DCless-H6-(C)ApoBL (Supporting Information, Figures S1 and S2), identical to the previously described ONCDCless-H6-(P)GKY2023 and ONC-DCless-H6-(P)ApoBL28 except that the linkers between the carriers and the peptides contain an Asp-Cys sequence. The new fusion proteins released peptides with a cysteine residue at the N-terminus, (C)GKY20 and (C)ApoBL, with efficiency only slightly lower than those observed in the case of (P)GKY20 and (P)ApoBL from ONCDCless-H6-(P)GKY20 and ONC-DCless-H6-(P)ApoBL. Furthermore we produced an anticancer peptide named p53pAnt33-35 containing the C-terminal region of the p53 protein fused by its carboxy-terminal end to the cell penetrating peptide Penetratin.36-38 The peptide with the additional cysteine, (C)p53pAnt, was efficiently produced by selective hydrolysis of fusion proteins H7-ONC-DCless-(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt (Figures S3 and S4), containing carriers based on ONC-DCless and ketosteroid isomerase (KSI)39, respectively. Moreover, the recombinant peptide (C)GKY20 could be modified using thiol selective reagents with an efficiency of about 100%, thus demonstrating that no undesired modification of the N-terminal cysteine residue was present.

Results and Discussion Acid cleavage analysis of recombinant (M23L)-ONC During the development of the ONC-DCless-H6 carrier we found that two Asp-Cys sequences present in (M23L)-ONC, at positions 18-19 and 67-68, underwent acid catalyzed hydrolysis with high efficiency. As no quantitative determination of cleavage efficiency was 8


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performed on that occasion, preliminarily to the development of a strategy to prepare recombinant peptides with a cysteine residue at the N-terminus, we decided to determine the sensitivity of the two sequences to the acidic hydrolysis. To this purpose, denatured and reduced recombinant (M23L)-ONC (Figure 2A) was incubated at 60°C for 24 h at pH 2, following the optimized cleavage conditions previously determined.23 (M23L)-ONC was analyzed by RP-HPLC before (Figure 2B) and after (Figure 2C) the acidic cleavage. Mass spectrometry analyses (Table 1) were performed to determine the identity of the peaks observed in the HPLC chromatograms. Cleavage of bonds D18-C19 and D67-C68 should provide three ONC fragments M(1)/D18, C19/D67 and C68/C104 (named F1, F2 and F3, respectively, in Figure 2A). An incomplete hydrolysis of the two Asp-Cys sequences should provide the larger fragments M(1)/D67 and C19/C104 (named F1-F2 and F2-F3, respectively, Figure 2A). With the exception of fragment F1-F2, all the expected fragments were identified. Moreover, we identified several shorter fragments deriving from the cleavage of the four Asp-X sequences present in fragments F1 and F2 (two Asp-X sequences in each fragment, Figure 2A). We also identified fragment M(-1)/V17 (named F1a, Table 1) deriving from the unusual cleavage at the N-side of Asp-18 (bond V17-D18), and a fragment (named F1ox, Table 1) with a molecular weight 16 Da higher than that expected for fragment F1, presumably an oxidized form of F1. On the basis of the peak areas and the predicted extinction coefficients of the fragments (Table S1) we estimated the relative cleavage efficiency of the acid labile bonds. From the comparison between the corrected area of the peaks, we estimated that the cleavage efficiency of D67-C68 bond was about 75-80% i.e. only slightly lower than those previously found for other Asp-Pro sequences.23 In the case of D18-C19 no fragment containing an uncleaved bond was identified. Even assuming that fragments were present but below detection limit it could be concluded that the D18-C19 bond had been hydrolyzed with an 9



efficiency of at least 90%. Furthermore, the higher sensitivity of this bond to acidic hydrolysis with respect to D67-C68 bond could be due to the presence of three adjacent AspX sequences (D16VD18CD20N). As for the cleavage of the Asp-X sequences, in all the four cases we determined cleavage efficiencies lower than 11%. Therefore, it can be concluded that both Asp-Cys sequences in (M23L)-ONC, in spite of the different sequence context (RDVDCDNI and YLSDCNVT), show a sensitivity to acidic hydrolysis comparable to that of Asp-Pro sequences and considerably higher than that of other Asp-X sequences.

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Figure 2. Cleavage analysis of (M23L)-ONC fusion protein. A) Amino acid sequence of (M23L)-ONC protein. Aspartate residues D2, D16, D18, D20, D32 and D67 are highlighted (bold). The three main peptides, named F1 (blue), F2 (red) and F3 (green) obtained by acid cleavage of the two Asp-Cys sequences (D18-C19 and D67-C68) are underlined. B) RPHPLC analysis of purified (M23L)-ONC protein. C) RP-HPLC analysis of peptides from (M23L)-ONC acid cleavage; recombinant protein was incubated at pH 60°C for 24 h at pH 2. The main peaks eluted between 25-35 min (D) and 60-75 min (E) retention times are enlarged. The bars under the chromatogram highlight the retention time intervals in which F1 (blue bar), F2 (red bar) and F3 (green bar) peptides were found. RP-HPLC analyses were carried out on Jupiter 5u C18 300A column by gradient 1 (see Supporting Information). Chromatograms were recorded at 280 nm. Amino acid sequences of purified peptides are reported in Table 1.

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Table 1. Mass spectrometry analyses of peptides released by chemical cleavage of (M23L)-ONC protein. Aspartic acid residues are pointed out in grey. Peak number 4 5 2 5 3 1

8 9 8 7

a

b

c

2392.67

MWExp (Da) 2375.93 2376.94 2392.35

2261.58

2260.93

-0,65

2162.45

2162.15

-0.30

2002.26

2002.02

-0.24

5539.38 5321.15

5540.46 5541,78 5322.26

+ 0.90 +2.4 +1.10

3934.56

3933.59

-1.00

MWThe (Da) 2376.67

Peptides F1 MQDWLTFQKKHITNTRDVD F1(ox)d MQDWLTFQKKHITNTRDVD F1ae MQDWLTFQKKHITNTRDV F1b MQDWLTFQKKHITNTRD F1c WLTFQKKHITNTRDVD F2 CDNILSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSD F2a NILSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSD F2b KNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSD

∆Exp- The (Da) -0.74 +0.27 -0.32

6

F3 CNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC

4077.78

4078.94

+1.16

10

F2-F3 CDNILSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSD CNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC F2a-F3 NILSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSD CNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC

9599.15

9604.13

+4,98

9380.92

9387.80

+6,88

10

f

(M23L)-ONC

MQDWLTFQKKHITNTRDVDCDNILSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYK LKKSTNKFCVTCENQAPVHFVGVGSC a

Theoretical molecular mass. Experimental molecular mass. c ∆Exp- The. Comparison of experimental and theoretical molecular weights (MWExp -MWTheo). d F1 peptide oxidized form. Experimental molecular mass of 2392.35 Da is consistent with the additional presence of oxygen atom (F1 peptide theoretical molecular weight, 2376.67 Da + 16 Da = 2392.67 Da). e Fragment M(-1)/V17 deriving from the unusual cleavage at the N-side of Asp-18. f (M23L)-ONC: amino acid sequence of recombinant Onconase mutant M23L. Two DC cleavage sites are marked (bold, underlined); four DX sequences are pointed out in grey. b

12


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Recombinant system to produce a peptide with N-terminal cysteine residue: ONCDCless-H6-(C)GKY20 fusion protein On the basis of the results described above we decided to mutate the Asp-Pro sequence present at the end of the flexible linker (GTGDP) in the fusion protein ONCDCless-H6-(P)GKY20 to Asp-Cys without additional modifications of the linker sequence. The coding sequence for mutated ONC-DCless-H6-(C)GKY20 (Figure S1) was obtained by chemical synthesis as described in Material and Methods. As expected, ONC-DCless-H6-(C)GKY20 was produced as inclusion bodies in E. coli BL21(DE3) strain (Figure 3) with yields similar to those of ONC-DCless-H6-(P)GKY20 (84 mg/L and 150 mg/L of LB and TB cultures, respectively; Table S2).

Figure 3. Expression of ONC-DCless-H6-(C)GKY20 in TB. SDS-PAGE (15%) analysis of fusion protein. Lane 1: Gallus gallus lysozyme (14.3 kDa); lanes 2,7: molecular markers (812-20-30-45-60-100-220 kDa proteins); lane 3: cellular lysate of the induced TB culture; lanes 4-5: insoluble and soluble fractions after cell lysis, respectively; lane 6: inclusion bodies after Triton/urea treatment. ONC-P: Onconase/Peptide fusion protein.

Differently from the parent protein, ONC-DCless-H6-(C)GKY20 contains a free cysteine which could undergo several oxidation reactions, including formation of disulfides and conversion to sulfenic, sulfinic and sulfonic acids, during the purification procedure, therefore we evaluated two alternative purification strategies. The first and simplest strategy consisted in the maintenance of a reducing environment throughout the purification. The 13



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inclusion bodies from TB cultures, after a cleaning step (Figure 3), were dissolved in the denaturing buffer (50 mM Tris-HCl pH 7.4, containing 6 M guanidine-HCl) in the presence of 2 mM TCEP, a well known reducing agent with oxygen scavenging ability. Samples were purged with N2 to remove oxygen and incubated for 3 h at 37°C. After incubation, the solubilized and reduced protein, herein called ONC-DCless-H6-(C)GKY20-SH, was purified by IMAC in the presence of 2 mM TCEP. Pooled fractions containing purified ONC-DClessH6-(C)GKY20-SH (SDS-PAGE analysis, Figure 4A) were extensively dialyzed against 0.1 M acetic acid pH 3.0 at 4°C. No reducing agent was added during this step as the acidic pH and the low temperature are usually sufficient to prevent oxidation of free cysteine residue. The Ellman test performed at the end of the dialysis showed that 100% of the expected cysteine residues were present as free thiols while mass spectrometry analysis further confirmed protein identity (Table S3). The reduced protein was immediately used for the selective acidic hydrolysis procedure described in the next section. Alternatively, the cysteine residue in ONC-DCless-H6-(C)GKY20 was protected through the formation of a mixed disulfide with cysteamine (2-aminoethanethiol) prior purification, using a procedure previously developed by our group for the purification of ONC and its mutants.24 To this aim inclusion bodies were dissolved in a modification buffer containing

6

M

guanidine-HCl

and

a

large

molar

excess

of

cystamine

(2-

aminoethanedisulfide). After incubation at 37°C, the sample was extensively dialyzed against ammonium acetate pH 5.0 to remove excess reagents. As highlighted by SDS-PAGE analysis (Figure 4B) the removal of guanidine-HCl at pH 5.0 led to precipitation of more than 95% of the fusion protein, whereas most of the E. coli contaminant proteins were found in the soluble fraction, thus allowing a first purification step.

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Figure 4. Purification and modification of ONC-DCless-H6-(C)GKY20. A) SDS-PAGE (15%) analysis of purified reduced ONC-DCless-H6-(C)GKY20-SH. Lane 1: molecular markers (8-12-20-30-45-60-100-220 kDa proteins); lane 2: Gallus gallus lysozyme (14.3 kDa); lane 3: ONC-DCless-H6-(C)GKY20-SH protein purified by IMAC (ONC-P/SH). B) SDS-PAGE (15%) analysis of modified ONC-DCless-H6-(C)GKY20-CEA protein after reaction with cystamine to obtain the protein with CEA-mixed disulfides (ONC-P/CEA). Lane 1: Gallus gallus lysozyme (14.3 kDa); lane 2: inclusion bodies after Triton/urea treatment; lanes 3-4: soluble and insoluble fractions after CEA-modification and dialysis against 50 mM AmAc buffer, pH 5, respectively. C) SDS-PAGE (15%) analysis of ONCDCless-H6-(C)GKY20-CEA (ONC-P/CEA) after IMAC purification. Lane 1: molecular 15



markers (8-12-20-30-45-60-100-220 kDa proteins); lane 2: Gallus gallus lysozyme (14.3 kDa); lane 3: ONC-DCless-H6-(C)GKY20-CEA protein purified by IMAC.

The insoluble fraction was collected and dissolved in 6 M guanidine-HCl. The Ellman test showed that only about 1.4% of the cysteine residues were present as free thiols, thus indicating that the yield of the modification reaction was close to 100%. The molecular mass of the modified protein (Table S3), herein called ONC-DCless-H6-(C)GKY20-CEA, was found to be 16033.22 Da, in good agreement with that expected for the fusion protein with a mixed disulfide with cisteamine (theoretical molecular weight 16034.55 Da). The protected fusion protein was purified by IMAC to homogeneity, as shown by the SDS-PAGE analysis (Figure 4C), and extensively dialyzed against 0.1 M acetic acid pH 3.0 at 4°C. The Ellman test allowed to determine that only about 2.5% of the cysteine residues were present as free thiols. The dialyzed protein was either immediately used for the selective acidic hydrolysis procedure or lyophilized and stored at -20 °C until use. We want to underline that we chose cystamine for our previous experience with this reagent, however it is reasonable to expect that other similar reagents could work with similar efficiency.

Chemical cleavage of ONC-DCless-H6-(C)GKY20 Release of (C)GKY20 from ONC-DCless-H6-(C)GKY20-SH was studied as function of pH (2 and 3), temperature (60°C and 70°C) and time (0-72 h) in the presence of TCEP as reducing agent. Determination of the percentage of cleaved protein by densitometry scanning of SDS-PAGEs (Figures S5 and S6) indicated that at 60°C (Figure 5) cleavage was slower at pH 3, with 80% of cleaved protein after 72 h, and faster at pH 2 with about 70-80% of cleaved protein after 24 h. Increasing temperature to 70°C led to cleavage percentages of about 60% at pH 3 and 70% at pH 2 after 12-15 h incubation, whereas 90% cleavage was observed after 72 h incubation. However, SDS-PAGE analyses (Figures S5 and S6) also 16


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showed that long incubation times (>48 h), especially at 70°C, caused the appearance of a smear below the band of the cleaved protein likely caused by non-specific hydrolysis. From these results we concluded that cleavage at 60°C and pH 2 for 24-36 h is a good compromise between yield of the cleavage products and absence of unwanted side-products.

Figure 5. Cleavage optimization of ONC-DCless-H6-(C)GKY20-SH. Hydrolysis of fusion protein was studied as function of pH (2 and 3), temperature (60°C and 70°C) and time (0-72 h). Percentage of cleaved protein was calculated by SDS-PAGE densitometry scanning as reported in Materials and Methods.

To investigate the influence of the oxidation state of the cysteine residue on the cleavage reaction, ONC-DCless-H6-(C)GKY20-CEA was incubated for 24 h at 60°C and pH 2 in presence and the absence of TCEP. SDS-PAGE analysis (Figure 6) showed that the protected protein in the presence of TCEP was hydrolyzed with an efficiency of about 80%, whereas, in the absence of TCEP it was hydrolyzed with an efficiency of about 5%. These findings confirm that efficient hydrolysis requires a reduced cysteine residue.

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Figure 6. Cleavage analysis of ONC-DCless-H6-(C)GKY20. Purified recombinant proteins were incubated at 60°C for 24 h at pH 2 in the absence and in the presence of TCEP reducing agent, and analyzed by SDS-PAGE (20 %). Lane 1: molecular markers (8-12-20-30-45-60100-220 kDa proteins); lanes 2,5: purified reduced (ONC-P/SH) and mixed disulfide (ONCP/CEA) proteins, respectively; lanes 3-4: reduced protein (ONC-P/SH) cleaved in the absence (lane 3) and in the presence (lane 4) of TCEP reducing agent; lanes 6-7: mixed disulfide protein (ONC-P/CEA) cleaved in the absence (lane 6) and in the presence (lane 7) of TCEP reducing agent; lane 8: GKY20 synthetic peptide. ONC-P: Onconase/Peptide fusion protein; ONC: Onconase carrier; P: (C)GKY20 peptide.

In the presence of TCEP, ONC-DCless-H6-(C)GKY20-SH and ONC-DCless-H6(C)GKY20-CEA were hydrolyzed with very similar efficiency, therefore the two alternative purification strategies can be considered equivalent. However, it should be noted that protection of the cysteine residue could allow a longer and safer storage of the protein. Lyophilized ONC-DCless-H6-(C)GKY20-CEA was stored at -20°C for at least six months without any decrease in the cleavage efficiency (data not shown).

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Preparation

and

hydrolysis

of

ONC-DCless-H6-(C)ApoBL,

H7-ONC-DCless-

(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt proteins To verify the general applicability of our strategy based on the cleavage of Asp-Cys sequences we prepared two further peptides with an N-terminal cysteine, namely (C)ApoBL and (C)p53pAnt, by cleavage of three different fusion proteins. ApoBL is an antimicrobial peptide previously prepared with an additional proline, (P)ApoBL, by cleavage of the fusion protein ONC-DCless-H6-(P)ApoBL.28 Therefore, as described above for (C)GKY20, we changed the Asp-Pro sequence to Asp-Cys thus obtaining the fusion protein ONC-DCless-H6-(C)ApoBL (Figure S2). p53pAnt is a well known 37 amino acid long anticancer peptide33-35, previously produced in E. coli in an insoluble form, fused to ketosteroid isomerase (KSI).39 The KSIp53pAnt fusion contained a thrombin site to release the peptide and a N-terminal seven histidine-tag (six histidine residues from a conventional H6-tag plus the N-terminal histidine residue of KSI). In this case we designed two fusion proteins named H6-KSI-DCless(C)p53pAnt (Figure S4) and H7-ONC-DCless-(C)p53pAnt (Figure S3). H6-KSI-DCless-(C)p53pAnt has two main differences with respect to the fusion protein described in Rodriguez and coworkers:39 six aspartate residues were mutated to glutamate to make KSI protein more resistant to acid hydrolysis as previously described for carrier ONC-DCless; the thrombin site between KSI and p53pAnt (CQAGPR/G) was replaced with an Asp-Cys site (SQGSD/CG). The single cysteine residue upstream the thrombin site was mutated to serine to leave a single cysteine residue in the fusion protein. H7-ONC-DCless-(C)p53pAnt contains a ONC-DCless carrier, like ONC-DCless-H6(C)GKY20 and ONC-DCless-H6-(C)ApoBL, but a seven-histidine tag was added to the Nterminus in place of the internal his tag to mimic the sequence at the N-terminal end of H6KSI-DCless-(C)p53pAnt. 19



Figure 7A highlights the four different sequences flanking the Asp-Cys sites in ONCDCless-H6-(C)GKY20, ONC-DCless-H6-(C)ApoBL, H7-ONC-DCless-(C)p53pAnt and H6KSI-DCless-(C)p53pAnt. Recombinant proteins were expressed in E. coli BL21(DE3) transformed with the corresponding pET22b(+) expression vectors (Figures S1-S4). As shown by SDS-PAGE analyses (Figure S7), all the proteins were expressed as inclusion bodies with high expression yields. ONC-DCless-H6-(C)ApoBL showed expression levels even higher than those of ONC-DCless-H6-(C)GKY20 (about 150 mg and 90 mg, respectively, per liter of LB culture), whereas, H7-ONC-DCless-(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt showed lower expression levels, i.e. about 60 mg and 70 mg of proteins per liter of LB culture, respectively, possibly due to a higher toxicity of p53pAnt with respect to GKY20 and ApoBL. Reduced proteins were purified by IMAC (Figure S7) carried out in the presence of TCEP and then dialyzed towards 0.1 M acetic acid pH 3.0 at 4°C as previously described for ONC-DCless-H6-(C)GKY20-SH, with high yields (Table S2). As in the case of ONCDCless-H6-(C)GKY20 the three recombinant proteins were fully reduced, as assessed by Ellman test. Furthermore, mass spectrometry analyses confirmed protein identities (Table S3). Chemical cleavage was carried out at 60°C and pH 2 in the presence of TCEP for incubation times up to 72 h. The percentages of cleaved fusion protein at each incubation time were assessed by densitometry analyses of SDS-PAGEs (Figure S8). As shown in Figure 7B, all the proteins showed cleavage kinetics similar to that of ONC-DCless-H6(C)GKY20-SH, thus suggesting that the sensitivity to the hydrolytic cleavage of Asp-Cys sequences is scarcely influenced by the nature of the flanking sequences. As in the case of ONC-DCless-H6-(C)GKY20-SH longer incubation times (> 48 h) led to high cleavage efficiency (about 90%) but also to a significant unspecific hydrolysis as 20


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evidenced by the presence of a smear below the bands of the cleaved carriers (Figure S8), therefore, routine cleavage was performed incubating the fusion proteins for 24 h (Figure S9A).

Figure 7. Cleavage analysis of fusion proteins. (A) Comparison of the sequences flanking the Asp-Cys site in (from top to bottom) ONC-DCless-H6-(C)GKY20, ONC-DCless-H6(C)ApoBL, H7-ONC-DCless-(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt. Violet, DC cleavage site; black, grey, and brown, C-terminal regions of the carriers ONC-DCless-H6, H7-ONC-DCless and H6-KSI-DCless, respectively; blue, green, and red, N-terminal regions of the peptides GKY20, ApoBL and p53pAnt, respectively. (B) Time-course of hydrolysis reaction carried out at 60°C, pH 2. Percentage of cleaved protein was calculated by SDSPAGE densitometry scanning as reported in Materials and Methods. Blue: ONC-DCless-H6(C)GKY20 [ONC-(C)GKY20]; green: ONC-DCless-H6-(C)ApoBL [ONC-(C)ApoBL]; red: H7-ONC-DCless-(C)p53pAnt [H7-ONC-(C)p53pAnt]; black: H6-KSI-DCless-(C)p53pAnt [H6-KSI-(C)p53pAnt].

21



Purification of (C)GKY20, (C)ApoBL and (C)p53pAnt To purify recombinant peptides from hydrolysis mixtures, we evaluated the possibility to selectively precipitate the carrier and the uncleaved protein at pH 7.2, a procedure successfully applied to purification of (P)GKY20 and several other peptides.23,28 To this purpose, pH was increased to 7.2 by dropwise addition of aqueous NH3 in the presence of 2 mM TCEP to prevent cysteine oxidation. Samples were incubated 1 h at 28°C, and the soluble and insoluble fractions were separated and analyzed by SDS-PAGE (Figure S9B). By spectral analyses of soluble fractions, we estimated that 100% of (C)ApoBL, 91% of (C)p53pANT derived from the cleavage of H7-ONC-DCless-(C)p53pAnt but only 36% of (C)GKY20 and 41% of (C)p53pANT derived from the cleavage of H6-KSI-DCless(C)p53pAnt were in the supernatant (Table S2). These data demonstrate that the effectiveness of the selective precipitation step strongly depends both on the carrier and the peptide. Based on these findings, preparative purification of (C)ApoBL and (C)p53pAnt from the ONC-DCless carrier was performed by selective precipitation at pH 7.2, whereas purification of (C)GKY20 and (C)p53pAnt from the KSI derived carrier was performed by direct loading of hydrolysis mixture on a C18 column. Peptides were purified with final yields (per liter of LB) of about 5 mg in the case of (C)GKY20, 16 mg in the case of (C)ApoBL, 5 mg in the case of (C)p53pAnt from H7-ONCDCless-(C)p53pAnt and 6 mg in the case of (C)p53pAnt from H6-KSI-DCless-(C)p53pAnt (Table S2). Expression in TB provided a higher final yield of (C)GKY20 (about 6-8 mg per liter of culture, Table S2). Purity of peptides, assessed by RP-HPLC (Figures S10-S11), typically ranged from 95% to 99%. The Ellman test and mass spectrometry analyses confirmed peptide identities and showed that all the cysteine residues were present in the reduced form and that no undesired modified form (e.g. oxidized peptides) was present in the sample (Table S3). 22


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Modification of (C)GKY20 Even if Ellman assay and mass spectrometry confirmed the presence of a free unmodified cysteine residue in the purified (C)GKY20, we decided to verify if the recombinant peptide possessed the expected reactivity toward a cysteine-specific alkylating agent, the thiol reactive fluorescent dye PyMPO maleimide, and CBT, a reagent specific for modification of 1,2-aminotiols. The two modified peptides, PyMPO-(C)GKY20 and LucGKY20 (Luc: luciferin moiety bound to GKY20) respectively, were obtained with yields of 100% (details can be found in Supporting Information, section S1, Figures S12-S15) thus demonstrating that purified (C)GKY20 has the expected reactivity. Furthermore, we also verified that the fluorescent label does not affect the antimicrobial activity of PyMPO(C)GKY20 (Supporting Information, section S2 and Figure S16).

Conclusions In this work we have described a new recombinant method to produce peptides with N-terminal cysteine residue as a convenient alternative to the chemical synthesis. The method is based on the release of the desired peptide from a suited carrier by chemical cleavage of an Asp-Cys sequence under mild reaction conditions. Our system is suitable for production of toxic peptides by using a carrier which drives the peptide into inclusion bodies. In addition to the previously reported carrier, ONC-DCless-H6, we have developed two further carriers optimized for the selective hydrolysis of Asp-Cys sites, i.e. H7-ONC-DCless and H6-KSIDCless. Even if these carriers were selected for their ability to drive toxic peptides into inclusion bodies, it is likely that they could be used for the expression of other peptides as well.

23



We have shown that cleavage of Asp-Cys sequences in many respects is similar to that of Asp-Pro sequences, in fact, both types of sequence can be selectively hydrolyzed in the same mild conditions, i.e. 60°C in aqueous acid at pH 2, and with similar efficiencies (7085% and 85-95%, respectively). Moreover, the two natural Asp-Cys sites in onconase and the four sites in the recombinant fusion proteins here described were hydrolyzed with similar efficiency, thus, it can be confidently hypothesized that Asp-Cys sequences will find wide applications in recombinant technologies like already demonstrated for Asp-Pro sites. Very interestingly, our data indicate that Asp-Cys sequences can be cleaved only when the Cys residue is reduced. The implications at the mechanistic level are yet to be studied, however, this observation suggests the possibility to control the hydrolysis of AspCys sequences by switching on and off sensitivity to acidic hydrolysis. We also developed two different protein purification strategies, one entirely carried out under reducing conditions, and the other in which cysteine thiol groups are protected as mixed disulfide, thus allowing a safer long term storage of the fusion protein. Both methods allowed the purification of (C)GKY20 with similar yields (6-8 mg/L of TB culture). Mass spectrometry analyses and the Ellman test of the peptides, as well as chemical modification of the N-terminal cysteine residue in (C)GKY20, demonstrated that the method here described is efficient and reliable allowing to prepare high purity peptides without unwanted modifications of the N-terminal cysteine residue.

Materials and Methods Plasmids DNA sequence coding for ONC-DCless-H6-(C)GKY20 fusion protein was obtained by chemical synthesis (MWG-Biotech AG; Ebersberg, Germany). All codons were optimized for expression in E. coli and restriction sites NdeI and SacI were introduced at 5’- and 3’-end 24


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of the synthetic gene, respectively, for its cloning into pET22b(+) vector (Figure S1). E. coli strain TOP10F’ was used for cloning procedure. Sequence of recombinant plasmid, named pET22b(+)/ONC-DCless-H6-(C)GKY20, was confirmed by DNA sequencing. pET22b(+) expression vectors coding for ONC-DCless-H6-(C)ApoBL, H7-ONCDCless-(C)p53pAnt

and

H6-KSI-DCless-(C)p53pAnt

pET22b(+)/ONC-DCless-H6-(C)ApoBL (C)p53pAnt

(Figure

S3)

and

(Figure

S2),

fusion

proteins,

named

pET22b(+)/H7-ONC-DCless-

pET22b(+)/H6-KSI-DCless-(C)p53pAnt

(Figure

S4),

respectively, were purchased from GenScript (USA Inc., Piscataway, NJ, USA). All codons were optimized for expression in E. coli.

Expression of recombinant proteins (M23L)-ONC, ONC-DCless-H6-(C)GKY20, ONC-DCless-H6-(C)ApoBL, H7-ONCDCless-(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt proteins were produced in E. coli BL21(DE3) strain, transformed with pET22b(+)/(M23L)-ONC24, pET22b(+)/ONC-DClessH6-(C)GKY20,

pET22b(+)/ONC-DCless-H6-(C)ApoBL,

pET22b(+)/H7-ONC-DCless-

(C)p53pAnt and pET22b(+)/H6-KSI-DCless-(C)p53pAnt plasmids, respectively, following the protocol already described.23,24 Details of experimental procedure are described in Supporting Information (section S3).

Chemical cleavage of (M23L)-ONC Recombinant protein was purified as previously described24 with minor modifications reported in Supporting Information (section S4). Chemical cleavage of purified (M23L)-ONC protein (about 1.4 mg/mL) was carried out in 0.1 M acetic acid, pH 2.0 (by addition of diluted HCl), as already described.23 Tris(2-carboxyethyl)phosphine hydrochloride (TCEPHCl) was added as thiol-free reducing agent40 at 1 mM final concentration. Sample was 25



incubated for 24 h in a water bath under nitrogen atmosphere at 60°C. Purified (M23L)-ONC and the peptide mixture derived from its chemical cleavage were analyzed by reverse-phase high performance liquid chromatography (RP-HPLC) with a Jasco LC-4000 system equipped with PU-4086 semi-preparative pumps and MD-4010 photo diode array detector. Analyses were carried out on Jupiter 5u C18 300A column (250 x 4,6 mm, 5µm particle size) provided by Phenomenex (Torrance, California, USA). Solvents were 0.05% trifluoracetic acid (TFA) in water (solvent A) and 0.05% TFA in acetonitrile (solvent B). Samples were eluted by linear gradient 1 (Supporting Information, section S5). Identity of peptides released by (M23L)-ONC hydrolysis, was assessed by mass spectrometry analyses of recovered peaks. Cleavage percentages of Asp-X sites were determined by comparison of integrated peak areas. Details are described in Supporting Information (section S6).

Purification of the recombinant proteins Denatured and reduced ONC-DCless-H6-(C)GKY20, ONC-DCless-H6-(C)ApoBL, H7-ONC-DCless-(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt were prepared from 100 mg of inclusion bodies dissolved in 20 mL of denaturing buffer (50 mM Tris-HCl, pH 7.4, containing 5 M guanidine-HCl) in the presence of 2 mM TCEP as reducing agent. Sample was incubated on a rotary shaker at 37°C for 3 h under nitrogen atmosphere. Soluble fraction was collected by centrifugation at 18,000 x g for 60 min at 4°C and the reduced protein was purified by immobilized metal ion affinity chromatography (IMAC) as described elsewhere23 in the presence of 2 mM TCEP. Mixed disulfide denatured ONC-DCless-H6-(C)GKY20 was produced by using cysteamine (CEA) as thiol protecting group following the procedure already described24 with minor modifications. Briefly, ONC-DCless-H6-(C)GKY20 fusion protein (100 mg) was dissolved in 20 mL of modification buffer (0.2 M Tris-acetate, pH 8.4, containing 10 mM 26


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EDTA, 6 M guanidine-HCl and 0.5 M cystamine dihydrochloride salt, the disulfide of cysteamine) and incubated at 37°C for 24 h under nitrogen atmosphere. Soluble fraction was collected by centrifugation at 18,000 x g for 60 min at 4°C and the reaction was stopped by glacial acetic acid addition up to pH 5.0. Sample was extensively dialyzed against 50 mM ammonium acetate buffer (AmAc), pH 5.0, at 4°C. The insoluble fraction containing ONCDCless-H6-(C)GKY20-CEA modified protein was separated from soluble fraction by centrifugation at 18,000 x g for 60 min at 4°C and washed three times with 30 mL of 50 mM AmAc buffer, pH 5. Insoluble modified protein was dissolved in 20 mL of denaturing buffer (50 mM Tris-HCl, pH 7.4, containing 5 M guanidine-HCl) at 37°C for 3 h under nitrogen atmosphere. The recombinant protein was purified by immobilized metal ion affinity chromatography (IMAC) as already described.23 Purified proteins (4 mg/mL) were extensively dialyzed against 0.1 M acetic acid at 4°C and stored at -80°C under nitrogen atmosphere. Molar concentration of free thiol groups was assessed by the Ellman assay41,42 (Supporting Information, section S7). Identity of modified recombinant protein was verified by mass spectrometry analysis.

Optimization of chemical cleavage Reduced ONC-DCless-H6-(C)GKY20 was hydrolyzed at pH 3 (acetic acid 0.1 M) or pH 2 (acetic acid 0.1 M acidified to pH 2 by addition of diluted HCl), in the presence of 1 mM TCEP as reducing agent. Samples were incubated at 60°C or 70°C for 72 h and monitored by SDS-PAGE analyses over the time. Cleavage percentages were estimated by densitometry analysis of SDS-PAGEs using ImageJ software (available for free download at: http://rsb.info.nih.gov/ij/).43 Hydrolysis of ONC-DCless-H6-(C)GKY20-CEA protein was tested in the presence or absence of 1 mM TCEP reducing agent at 60°C, pH 2, for 24 h. Hydrolysis was monitored by 27



SDS-PAGE as described above. Formaldehyde was used to covalently link proteins and polypeptides in polyacrylamide gels.44

Large scale chemical cleavage of fusion proteins Release of peptides from fusion proteins was carried out as already described23 with minor modifications. Briefly, purified fusion proteins (about 2 mg/mL in 0.1 M acetic acid, pH 2.0 by addition of diluted HCl), were incubated for 24 h in a water bath under nitrogen atmosphere at 60°C, in the presence of 1 mM TCEP as reducing agent (6:1 ratio for TCEP over thiols). Cleavage efficiency was estimated by densitometry analysis of 20% SDS-PAGE. Formaldehyde was used to covalently link proteins and polypeptides in polyacrylamide gels.44 Percentage of cleaved protein was determined through image analysis with ImageJ software (available for free download at: http://rsb.info.nih.gov/ij/).43

Purification of recombinant peptides The pH of hydrolysis mixtures of ONC-DCless-H6-(C)ApoBL and H7-ONC-DCless(C)p53pAnt proteins was increased to pH 7.2 by adding diluted NH3 in the presence of 2 mM TCEP for 1 h at 28°C under nitrogen atmosphere. Insoluble fusion proteins and carriers were separated from soluble peptides by 30 min centrifugation at 18,000 x g at 4°C. Soluble (C)ApoBL and (C)p53pAnt were further purified by RP-HPLC on Europa Protein 300 C18 column (5µm, 25 x 1) from Teknokroma (Barcelona, Spain). Samples were loaded in 5% acetonitrile (0.1% TFA) at a flow rate of 2 mL/min and eluted by linear gradient 6 (Supporting Information, section S5). (C)GKY20 and (C)p53pAnt released from the hydrolysis of H6-KSI-DCless(C)p53pAnt were purified by RP-HPLC on Europa Protein 300 C18 column omitting the selective precipitation step. Hydrolysis mixtures were directly loaded in 5% acetonitrile 28


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(0.1% TFA) at a flow rate of 2 mL/min and eluted by linear gradients 2 and 6, respectively (Supporting Information, section S5). Purified peptides were lyophilized and dissolved in 5 mM AmAc buffer, pH 5.0. Samples were stored at -80°C under nitrogen atmosphere. Molar concentration of free thiol groups was assessed by the Ellman assay (Supporting Information, section S7).41 Purity of peptides was evaluated by SDS-PAGE (20%) and RP-HPLC on Jupiter 5u C18 300A column and Europa Protein 300 C18 column. Peptides were eluted by linear gradients 3-4-6 (Supporting Information, section S5). Identity of peptides was confirmed by mass spectrometry analyses.

Peptide Labeling Purified (C)GKY20 peptide (0.5 mg) was labeled by the thiol reactive fluorescent dye PyMPO maleimide (1-[2-(Maleimido)ethyl]-4-[5-(4-methoxyphenyl)-2-oxazolyl]pyridinium triflate),

and

by

a

site-specific

N-terminal

modification

reagent,

6-hydroxy-2-

cyanobenzothiazole (CBT). Experimental details are reported in Supporting information file (section S8).

Mass spectrometry analyses Analyses were performed on MALDI-TOF micro MX spectrometer (Waters, Manchester, UK) and MALDI-TOFTOF 4800 (Applied Biosystems, Framingham, MA, USA) instrument. Experimental details are reported in Supporting Information (section S9).

Antimicrobial assay

29



Antibacterial activity assays were carried out by plate viable-count method performed as previously described.23,45 Experimental details are reported in Supporting Information (section S10).

Materials and general procedures Details of chemicals, molecular biology materials and general methods are reported in Supporting Information (sections S11-S12).

Acknowledgments This work was supported by grants FFC#12/2014, and FFC#20/2014 from “Italian Cystic Fibrosis Research Foundation” (http://www.fibrosicisticaricerca.it), by grant “Programma F.A.R.O IV tornata” (CUP: E68C12000260003), co-funded by Polo delle Scienze e Tecnologie, Università di Napoli Federico II and “Compagnia di San Paolo”, and by Università di Napoli Federico II (CDA N. 19, 28/12/2016).

Supporting Information Additional experimental procedures including protein expression and purification, RP-HPLC and Mass spectrometry analyses, cleavage percentage determination of Asp-X sites, peptide labeling, antimicrobial assay by plate viable-count method, supplementary figures and tables, gene and protein sequences. (PDF)

Abbreviations ONC, onconase; KSI, ketosteroid isomerase; GKY20, human thrombin antimicrobial C-terminal peptide; (C)ApoBL,, antimicrobial peptide from an inner region of human Apolipoprotein B;

(C)p53pAnt, anticancer peptide containing the C-terminal region of the p53 protein fused to 30


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the

cell

penetrating

peptide

Penetratin;

methoxyphenyl)-2-oxazolyl]pyridinium triflate);

PyMPO,

(1-[2-(Maleimido)ethyl]-4-[5-(4-

CBT, 6-hydroxy-2-cyanobenzothiazole;

Luc-GKY20, Luciferin linked GKY20 peptide; TCEP-HCl, Tris(2-carboxyethyl)phosphine hydrochloride.

31



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Table of Contents (TOC)

37



Figure 1. Examples of reactions for cysteine modification. Panel A shows three popular reactions for the modification of terminal or internal cysteine residues: formation of a mixed disulfides (1) alkylation by substituted maleimides (2), alkylation by substituted haloacetamides (3). Panel B shows three reactions typical of N-terminal cysteine residues: reaction with thioesters (4), cyclization with aldehydes (5), cyclization with 2-cyanobenzothiazoles (6). P1 can be a hydrogen atom or the polypeptide chains upstream to the cysteine residue. P2 can be a OH group or the polypeptide chain downstream to the cysteine residue. R1 and R2 can be a wide variety of groups including labels, fluorophores, protein/peptides and solid supports. R3 usually is a small alkyl or aryl group. EW is an electron withdrawing group (e.g. the nitrobenzoate group in the Ellman reagent). X is iodide or bromine. Cysteine bonds and atoms are shown in bold for clarity. 109x154mm (300 x 300 DPI)


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Figure 2. Cleavage analysis of (M23L)-ONC fusion protein. A) Amino acid sequence of (M23L)-ONC protein. Aspartate residues D2, D16, D18, D20, D32 and D67 are highlighted (bold). The three main peptides, named F1 (blue), F2 (red) and F3 (green) obtained by acid cleavage of the two Asp-Cys sequences (D18C19 and D67-C68) are underlined. B) RP-HPLC analysis of purified (M23L)-ONC protein. C) RP-HPLC analysis of peptides from (M23L)-ONC acid cleavage; recombinant protein was incubated at pH 60°C for 24 h at pH 2. The main peaks eluted between 25-35 min (D) and 60-75 min (E) retention times are enlarged. The bars under the chromatogram highlight the retention time intervals in which F1 (blue bar), F2 (red bar) and F3 (green bar) peptides were found. RP-HPLC analyses were carried out on Jupiter 5u C18 300A column by gradient 1 (see Supporting Information). Chromatograms were recorded at 280 nm. Amino acid sequences of purified peptides are reported in Table 1. 129x152mm (300 x 300 DPI)


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Figure 3. Expression of ONC-DCless-H6-(C)GKY20 in TB. SDS-PAGE (15%) analysis of fusion protein. Lane 1: Gallus gallus lysozyme (14.3 kDa); lanes 2,7: molecular markers (8-12-20-30-45-60-100-220 kDa proteins); lane 3: cellular lysate of the induced TB culture; lanes 4-5: insoluble and soluble fractions after cell lysis, respectively; lane 6: inclusion bodies after Triton/urea treatment. ONC-P: Onconase/Peptide fusion protein. 70x53mm (300 x 300 DPI)


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Figure 4. Purification and modification of ONC-DCless-H6-(C)GKY20. A) SDS-PAGE (15%) analysis of purified reduced ONC-DCless-H6-(C)GKY20-SH. Lane 1: molecular markers (8-12-20-30-45-60-100-220 kDa proteins); lane 2: Gallus gallus lysozyme (14.3 kDa); lane 3: ONC-DCless-H6-(C)GKY20-SH protein purified by IMAC (ONC-P/SH). B) SDS-PAGE (15%) analysis of modified ONC-DCless-H6-(C)GKY20-CEA protein after reaction with cystamine to obtain the protein with CEA-mixed disulfides (ONC-P/CEA). Lane 1: Gallus gallus lysozyme (14.3 kDa); lane 2: inclusion bodies after Triton/urea treatment; lanes 3-4: soluble and insoluble fractions after CEA-modification and dialysis against 50 mM AmAc buffer, pH 5, respectively. C) SDS-PAGE (15%) analysis of ONC-DCless-H6-(C)GKY20-CEA (ONC-P/CEA) after IMAC purification. Lane 1: molecular markers (8-12-20-30-45-60-100-220 kDa proteins); lane 2: Gallus gallus lysozyme (14.3 kDa); lane 3: ONC-DCless-H6-(C)GKY20-CEA protein purified by IMAC. 70x180mm (300 x 300 DPI)




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Figure 5. Cleavage optimization of ONC-DCless-H6-(C)GKY20-SH. Hydrolysis of fusion protein was studied as function of pH (2 and 3), temperature (60°C and 70°C) and time (0-72 h). Percentage of cleaved protein was calculated by SDS-PAGE densitometry scanning as reported in Materials and Methods. 80x75mm (300 x 300 DPI)



Figure 6. Cleavage analysis of ONC-DCless-H6-(C)GKY20. Purified recombinant proteins were incubated at 60°C for 24 h at pH 2 in the absence and in the presence of TCEP reducing agent, and analyzed by SDSPAGE (20 %). Lane 1: molecular markers (8-12-20-30-45-60-100-220 kDa proteins); lanes 2,5: purified reduced (ONC-P/SH) and mixed disulfide (ONC-P/CEA) proteins, respectively; lanes 3-4: reduced protein (ONC-P/SH) cleaved in the absence (lane 3) and in the presence (lane 4) of TCEP reducing agent; lanes 6-7: mixed disulfide protein (ONC-P/CEA) cleaved in the absence (lane 6) and in the presence (lane 7) of TCEP reducing agent; lane 8: GKY20 synthetic peptide. ONC-P: Onconase/Peptide fusion protein; ONC: Onconase carrier; P: (C)GKY20 peptide. 80x58mm (300 x 300 DPI)


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Figure 7. Cleavage analysis of fusion proteins. (A) Comparison of the sequences flanking the Asp-Cys site in (from top to bottom) ONC-DCless-H6-(C)GKY20, ONC-DCless-H6-(C)ApoBL, H7-ONC-DCless-(C)p53pAnt and H6-KSI-DCless-(C)p53pAnt. Violet, DC cleavage site; black, grey, and brown, C-terminal regions of the carriers ONC-DCless-H6, H7-ONC-DCless and H6-KSI-DCless, respectively; blue, green, and red, N-terminal regions of the peptides GKY20, ApoBL and p53pAnt, respectively. (B) Time-course of hydrolysis reaction carried out at 60°C, pH 2. Percentage of cleaved protein was calculated by SDS-PAGE densitometry scanning as reported in Materials and Methods. Blue: ONC-DCless-H6-(C)GKY20 [ONC-(C)GKY20]; green: ONC-DCless-H6-(C)ApoBL [ONC-(C)ApoBL]; red: H7-ONC-DCless-(C)p53pAnt [H7-ONC-(C)p53pAnt]; black: H6-KSI-DCless-(C)p53pAnt [H6-KSI-(C)p53pAnt]. 80x98mm (300 x 300 DPI)



199x130mm (300 x 300 DPI)


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Chemical Cleavage of an Asp-Cys Sequence Allows Efficient

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