Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Proteome-based identification of signal peptides for improved secretion of recombinant cyclomaltodextrin glucanotransferase in Escherichia coli How Lie Linga, Zaidah Rahmatb, Abdul Munir Abdul Muradc, Nor Muhammad Mahadic,d, ⁎ Rosli Md. Illiasa, a

Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia c School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia d Malaysia Genome Institute, Ministry of Science, Technology and Innovation Malaysia, Jalan Bangi, 43000 Kajang, Selangor, Malaysia b

A R T I C L E I N F O

A B S T R A C T

Keywords: Proteome Signal peptide Protein secretion

Secretion of recombinant proteins in heterologous host has drawn attention for its simpler purification and downstream processes. Searching for secretion aid molecules to improve protein secretion can be done through synthetic biology, screening of genome data and proteome-based approach. In the present study, the extracellular proteome on starch-containing medium of Bacillus lehensis G1 was analyzed to identify naturally secreted proteins with signal peptide. A total of 87 protein spots were identified by mass spectrometry, which were categorized mostly in the metabolism of carbohydrates and related molecules (20%). Over-expression and secretion studies were performed for all the 14 selected signal peptides fused to a reporter protein, cyclomaltodextrin glucanotransferase (CGTase). All clones were found to allow CGTase to be excreted into the medium, as observed and measured from the iodine plate assay and enzyme activity assay. Compared to native signal peptide (G1) of CGTase, signal peptide of GlcNAc-binding protein A (GAP) significantly improved CGTase activities by 735% and 205% in extracellular and periplasmic compartment, respectively, with an increase of only ∼1.7 fold the amount of β-galactosidase (cell lysis) in the medium. GAP has the highest secretion rate of 45.6 U/ ml/h among all clones, where physicochemical characteristics of signal peptide play significant role.

1. Introduction In modern biotechnology industries, recombinant protein production in microbial cell is an attractive research. With the understanding of the bacterial protein translocation mechanism, efforts have been made to target recombinant proteins to the translocation machinery and release it to the extracellular milieu. Secretion of heterologous proteins is used as a natural separation step for easier harvesting, simpler downstream purification by avoiding contamination with intracellular proteins [1], and preventing misfolding and aggregation which caused inclusion bodies formation. Escherichia coli (E. coli) has a well-studied genetics background, fast growth rate and high production level which can be achieved in a costeffective way [2]. However, E. coli does not naturally secrete proteins into the extracellular medium under standard laboratory conditions [3]. Therefore, it is a challenge to engineer E. coli in producing high level of secreted proteins with minimum occurrence of cell lysis [4]. Various attempts have been devised to improve protein secretion in E.

⁎

coli such as modification of cultivation conditions [5], use of dedicated secretion system that naturally exists in E. coli [6] and manipulation of heterologous signal peptides [7]. E. coli and Bacillus species shared similar features, including the use of signal peptide in protein transportation mechanisms even though they have different cell membrane and cell wall structures. The use of heterologous signal peptides from Bacillus species can be recognized by the E. coli secretion machinery and resulted in high yields of secreted proteins in the periplasmic and extracellular space when expressed in E. coli [8]. A signal peptide contains information needed to direct desired protein to the translocation pathway thus has great influence in protein secretion. Periplasmic localization of maltogenic amylase (MA) was enhanced when fused to codon-optimized signal peptides compared to pectate lyase subunit (PelB) signal peptide [9]. In addition, extracellular expression of cutinase mediated by signal peptide was prompted by cutinase’s phospholipid hydrolase activity [10]. Several examples of the use of Bacillus sp. signal peptides in E. coli host such as secretion of human leptin [11] were reported. Signal peptides from

Corresponding author. E-mail address: [email protected] (R.Md. Illias).

http://dx.doi.org/10.1016/j.procbio.2017.06.018 Received 6 February 2017; Received in revised form 1 June 2017; Accepted 6 June 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Ling, H.L., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.06.018

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2.2. Preparation of extracellular proteins for proteome analysis

Bacillus sp. were proven to work well in E. coli expression system. Although signal peptide could function in cross-host, it is interesting to note that, there is a need for individually optimal signal peptides for every recombinant protein secretion [12]. The translocation efficiency of a desired protein is securely linked to its compatibility with the signal peptide used and does not promised high secretion production [1]. Therefore, several attempts have been developed in finding potential signal peptide to improve extracellular protein production. These attempts include the mutation of signal peptide [13]; codon-optimization of signal peptide [9]; systematic screening of genomic data for signal peptide [12]; the use of synthetic signal peptide [14] and proteomebased approach [15]. Previously, CGTase from Bacillus sp. G1 was cloned and fused with its native signal peptide (G1) for expression in E. coli and the enzyme was successfully secreted to the extracellular space [16]. Mutated G1 signal peptide (i.e. M5) showed a significant increase in extracellular CGTase which was about 1.9-fold higher but less cell lysis compared to E. coli signal peptide (i.e. pelB) [17]. Furtheremore, there were several reports on the use of E. coli signal peptides (i.e. PelB and OmpA) to improve CGTase secretion in E. coli expression system [18–20]. However, low yield (5.83 U/ml) of CGTase in the extracellular space was obtained when using E. coli signal peptide [21]. Strategies such as medium additives and fed-batch cultivation are required in addition to presence of signal peptide for achieving higher CGTase yield in E. coli medium [22]. The use of signal peptide to export proteins to the extracellular space was reviewed by Shokri et al. [23] where the extracellular production of CGTase by the native Bacillus sp E1 signal peptide without cell lysis was successful in E. coli. The 87 kDa precursor protein was processed and secreted as mature enzyme of 81 kDa via periplasm to the extracellular space [24]. Lipase signal peptide from Bacillus subtilis has also been used to secrete recombinant CGTase into the culture medium of E. coli, representing 88% (3 U/ml of culture) of the total protein activity [25]. This shows a high potential for signal peptide originated from Bacillus sp. to work well in E. coli for enzyme expression. In our previous study, M5 signal peptide enhanced secretion of CGTase but still exhibited a high cell lysis rate where glycine supplementation is required to improve cell viability of E. coli [17]. We hypothesized that secretion of CGTase can be improved by fusing with other signal peptides from Bacillus sp. Hence, in this study, a highthroughput proteomic analysis of the extracellular proteins of B. lehensis G1 was conducted to allow identification of its associated signal peptides for use in excretory protein production in E. coli. The exoproteome of B. lehensis G1 was analyzed by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF). The fourteen potential signal peptides were evaluated for extracellular production of CGTase in E. coli. Using this approach, signal peptide of GlcNAc-binding protein A (GAP) was found as the optimal signal peptide that lead to significant increases in the amount of secreted CGTase in E. coli.

B. lehensis G1 extracellular proteins were collected at mid-log phase as previously described [26] with slight modification. Cells were removed from the growth medium via centrifugation at 10414 × g and 4 °C for 15 min. Proteins in the supernatant were precipitated with 10% (w/v) pre-chilled trichloroacetic acid (TCA) for 30 min and were collected via centrifugation at 10414 × g for 15 min. The resulting protein pellet was collected and washed twice with pre-chilled acetone. The supernatant was removed, and the resulting protein pellet was air-dried for 5 min. Finally, the pellet was resolubilized in rehydration buffer (8 M urea, 40 mM dithiotreitol (DTT), 2% CHAPS, 0.5% (v/v) carrier ampholytes, 1 mM protease inhibitor cocktail, 0.002% bromophenol blue). The protein concentration of the extracellular protein sample was determined using a 2-D Quant Kit (GE Healthcare, United Kingdom) according to the manufacturer’s protocols. 2.3. Two-dimensional gel electrophoresis (2-DE), gel analysis, and protein identification 1D isoelectric focusing (IEF) was carried out using an IEF 100 (Hoefer, United States) and 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad, United States) was conducted using a VS20 WAVE Maxi (Cleaver Scientific Ltd, United Kingdom). The protocols were carried out according to manufacturer recommendations. Protein spots were in-gel digested using a trypsin digestion kit (Thermo Scientific, United States). The digested peptides were purified and concentrated using ZipTip C18 (Merck Milipore, United States) before spotting onto a target plate (AnchorChip Standard, 800 um; Bruker, United States). An UltraFlex MALDI-TOF/TOF mass spectrometer (Bruker) was used to analyze the digested peptides. Mass spectrometry (MS) spectra were gathered with 3000 laser shots per spectrum, and tandem mass spectrometry (MS/MS) spectra were acquired with 4000 laser shots per fragmentation spectrum. The peptide mass fingerprinting (PMF) peaks with the highest mass intensities (maximum 20 strongest peaks) were selected as precursor ions to acquire MS/MS fragmentation data. Bruker Daltonics Bio tools 3.2 SR3 was used for spectra analyses and the generation of peak list files. The signal-to-noise threshold was set at 7. The peak list files were used to search an inhouse B. lehensis G1 database (4017 sequences; 1166855 residues) using MASCOT version 2.4 (Matrix Science). The search parameters were set for proteolytic enzymes: trypsin, one maximum missed cleavage, variable modification of oxidation (Methionine), fixed modification of cys residues carbamidomethylation and peptide mass tolerance for monoisotopic data of 100 ppm, and a fragment mass tolerance of 0.4 Da. 2.4. Construction of expression plasmids Bacterial strains used in this study and their relevant characteristics are described in Table 1, while oligonucleotide polymerase chain reaction (PCR) primers used to amplify genes are summarized in Table 2. The plasmid CG21 (plasmid pET21a(+) carrying CGTase gene) that allows the insertion of signal peptides upstream of mature CGTase gene through NheI and BamHI restriction sites was used as expression vector. Amplification of DNA was performed using KAPA HiFi HotStart ReadyMix PCR kit (Kapa Biosystems, United States). To study expression levels, plasmids were subsequently introduced into E. coli BL21(DE3) [F− ompT hsdSB (rB- mB-) gal dcm (DE3)] which was purchased from Novagen. All restriction enzymes and T4 DNA ligase were purchased from NEB, United States and Promega, United States, respectively.

2. Materials and methods 2.1. Culture media, antibiotics and incubation conditions B. lehensis G1 was cultured in Horikoshi medium supplemented with 1% (w/v) starch (Merck, Germany). E. coli BL21(DE3) was cultured at 37 °C in Luria-Bertani (LB) broth as expression host. Antibiotics (100 μg/ml ampicillin) were added when necessary. Cell growth was monitored by measuring the absorbance at 600 nm (OD600) with an Ultrospec 1100 pro spectrophotometer (Amersham Biosciences, Uppsala, Sweden). pET systems from Novagen were used as the vector backbones for cloning the heterologous signal peptides fused to the CGTase gene.

2.5. Signal peptide analysis Signal peptide prediction were computed using Phobius (http:// phobius.sbc.su.se/) and SignalP (http://www.cbs.dtu.dk/services/ 2

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Table 1 Bacterial strain and plasmids used in this study. Strain or plasmids Strain E. coli BL21(DE3) Plasmids pET21a(+) G1 GAP CSP 1853 1848 ECDS lytE Chit BGL BGL4 TNPP MEP 3542 3571

Relevant characteristics and use

Source

E. coli F− ompT hsdSB (rB- mB-) gal dcm (DE3)

Novagen, USA

5.4 kb, resistant to ampicilin, T7 promoter, pBR322 origin pET21a(+) derivative carrying CGTase containing G1 SP pET21a(+) derivative carrying CGTase containing GlcNAc-binding protein A signal peptide pET21a(+) derivative carrying CGTase containing Cell surface protein signal peptide pET21a(+) derivative carrying CGTase containing 1853 signal peptide pET21a(+) derivative carrying CGTase containing 1848 signal peptide pET21a(+) derivative carrying CGTase containing Endonuclease/CDSuclease/phosphatase signal peptide pET21a(+) derivative carrying CGTase containing Endopeptidase lytE signal peptide pET21a(+) derivative carrying CGTase containing Chitinase signal peptide pET21a(+) derivative carrying CGTase containing Endo-beta-1,3-glucanase signal peptide pET21a(+) derivative carrying CGTase containing Endo-1,3(4)-beta-glucanase 1 signal peptide pET21a(+) derivative carrying CGTase containing Trifunctional nucleotide phosphoesterase protein signal peptide pET21a(+) derivative carrying CGTase containing Minor extracellular protease signal peptide pET21a(+) derivative carrying CGTase containing 3542 signal peptide pET21a(+) derivative carrying CGTase containing 3571 signal peptide

Novagen, USA [17] This study This study This study This study This study This study This study This study This study This study This study This study This study

was inoculated in 5 ml of LB broth containing 100 μg/ml ampicillin and incubated at 37 °C for 16 h. Five-hundred microlitre of the overnight culture was inoculated into 50 ml of LB broth containing 100 μg/ml ampicillin and incubated at 37 °C until OD600nm reached 0.7. Recombinant gene expression was induced by addition of 0.01 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and further incubation at 30 °C for 24 h. Samples were collected at different time points postinduction to determine the profile of extracellular protein expression.

Table 2 Oligonucleotide PCR primers used in this study. Primer name

Sequence (5′−3′)a

FGAP RGAP FCSP RCSP F1853 R1853 F1848 R1848 FECDS RECDS FlytE RlytE FChit RChit FBGL RBGL FBGL4 RBGL4 FTNPP RTNPP FMEP RMEP F3542 R3542 F3571 R3571

GCGC GCTAGC TTG ATT GGT AAA AAG ATAT GGATCC TGC ACT TGC CGA TTC GCGC GCTAGC ATG AGT ATC CAA TGG AAT GCAT GGA TCC TGC CTT ACT AAC TGA TGG GCGC GCTAGC ATG AGG TAT CAG AAA GAA ATAT GGATCC TGC TAA AGC GGG TTG TTG GCGC GCTAGC ATG AAA AAA ATC AAA GTA ATAT GGATCC TGC TAA TGC CGT ATT CCC GCGC GCTAGC GTG AGA AAA GTG TTG AAA GCAT GGATCC GGC ATA TAC TTG CTT ATC GCGC GCTAGC TTG AGC AAA TCG AAT CTA ATAT GGATCC TGC TTC TGA CAC TTG TGG GCGC GCTAGC ATG AGA GTA AAA CGT ACT ATAT GGATCC TGC CCA TAG TGC TTG ATC GCGC GCTAGC ATG AAG AAA GTA ATA GGA ATAT GGATCC TGC ACC GGC ATA TGA AGT GCGC GCTAGC ATG AAA AAA AGA TGG TTC GCCT GGATCC TTG ATT GGC GAA ACT TGA GCGC GCTAGC ATG GAG AAA TGG AAA AAG ATAT GGATCC TGC TTT CGC CTC TCC ATT GCGC GCTAGC TTG AAA AAA ACG ACA GTAT GGATCC CGC TTT TAC GTA CTG GCGC GCTAGC ATG AAA AAA GCT TGT TCT GCAT GGATCC CGA TGA TGC GAA AGA ATA GCGC GCTAGC ATG AAA AAG GTA TTT AGC GCGT GGATCC TGC ATA AGC GTC TTT AGA

a

2.8. Cell fractionation One milliliter of culture was harvested, and the cells were pelleted by centrifugation at 10414 × g at 4 °C for 10 min. The supernatant fraction was collected. The cell pellet was resuspended in 490 μl 10 mM Tris-HCl, pH8.0 with 20% (w/v) sucrose, and EDTA was added to a final concentration of 10 mM. The cell suspension was incubated at 4 °C for 10 min and pelleted by centrifugation at 10414 × g at 4 °C for 10 min. The supernatant was collected into periplasmic tube collectors, and the cells were resuspended in 490 μl ice-cold 10 mM MgSO4 before at 4 °C for 10 min. The cells were pelleted by centrifugation as above, and the supernatant was kept with the first periplasmic sample to obtain a total periplasmic fraction. The pellet was resuspended in 200 μl Bugbuster (Novagen, USA) and 0.2 μl 10 mg/ml benzonase and 2 μl 10 mg/ml lysozyme were added into the cell suspension. The cell suspension was incubated at room temperature for 20 min with 200 rpm shaking. The supernatant were collected as a cytoplasmic fraction. Each fraction were analyzed using SDS-PAGE [27] followed by enzymatic activity assays.

Underlines indicate restriction sites: NheI, GCTAGC; BamHI, GGATCC.

SignalP/). Different physicochemical characteristics of the signal peptides including grand average of hydropathicity (GRAVY) were evaluated by ProtParam online tool at http://web.expasy.org/protparam/

2.9. Quantification of CGTase

CGTase activity was detected using iodine plate assay method. All strains were streaked on LB agar containing 100 μg/ml ampicillin, 1% starch and supplemented with 0.5 mM lactose as an inducer. The cultured plate was incubated overnight at 37 °C. Iodine test was performed by pouring 2 ml of 0.01 M I/KI solution onto the plates. The formation of halo zone indicated positive CGTase activity in the extracellular location.

CGTase activity was quantified using a phenolphthalein assay [17]. The reaction mixture containing 1 ml of 0.04 g starch in 0.1 M sodium phosphate buffer (pH 7.0) and 0.1 ml enzyme solution was incubated at 60 °C for 10 min in a water bath. The reaction was stopped by adding 3.5 ml of 0.03 M NaOH solution. A volume of 0.5 ml of 0.02% (w/v) phenolphthalein in 0.005 M Na2CO3 was then added to the reaction mixture. After 15 min incubation at room temperature, the decrease in color intensity was measure at 550 nm. One unit of enzyme activity was defined as the amount of enzyme that forms 1 μmol β-cyclodextrin/ min.

2.7. CGTase expression in recombinant hosts

2.10. Beta-galactosidase enzyme assay

2.6. Iodine plate assay

Assay of β-galactosidase was conducted as an indicator of cell lysis

Recombinant E. coli harboring the appropriate expression plasmids 3

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Fig. 1. The extracellular proteome of B. lehensis G1 cultured in Horikoshi medium at 37 °C. Extracellular proteins prepared as described in Materials and Methods section, were separated by 2-DE in pH range of 4–7. The identified proteins with signal peptide are labelled.

Table 3 Identified extracellular proteins of B. lehensis G1 with their predicted signal peptides. Signal peptide

Gene no.a

Protein descriptionb

Predicted locationc

Theoretical pI

Theoretical Mw (kDa)

Signal peptide amino acid sequenced

G1 GAP

AIC96492 AIC95922

Cyclomaltodextrin glucanotransferase GlcNAc-binding protein A

Extr Extr

4.72 7.25

78.62 28.95

CSP 1853 1848 ECDS LytE Chit BGL BGL(4) MEP

AIC95945 AIC94431 AIC94426 AIC92706 AIC96238 AIC93540 AIC93282 AIC96475 AIC95833

Cell surface protein Hypothetical protein, conserved Hypothetical protein, conserved Endonuclease/CDSuclease/phosphatase Endopeptidase lytE Chitinase Endo-beta-1,3-glucanase Endo-1,3(4)-beta-glucanase 1 Minor extracellular protease

Extr Extr Extr V Extr Extr Extr Extr Extr

4.83 4.32 4.66 4.37 5.37 4.42 4.33 4.53 4.1

24.64 72.67 26.55 33.96 52.44 62.12 31.64 99.76 83.88

TNPP

AIC95918

V

4.2

100.2

3542 3571

AIC96089 AIC96118

Trifunctional nucleotide phosphoesterase protein Hypothetical protein, conserved Hypothetical protein, conserved

LNDLNDFLK TISLSFIFF LLLSLPTVAEA LIGKKKGHSFLQSI GFTVLASALFVF ANAESASA MSIQWNK VCLLLGATFLFL SVVFPSVSKA MRYQKE FFATAMLSFMVL SLQQPALA MKKIK VLTITALAFALI TSGNTALA VRKVLK VGVTLAVLTSV SYLLSTDKQVYA LSKSNLNKF LFSSAVVAGVVA VAPQVSEA MRVKRT SLLLIVITLLFF NGSDQALWA MKK VIGALSIAACTTLF ATSYAGA MKKRWF WFVLALIVLFPF QSSFANQ LKKTTNLNCTK SLSVVALSFCMV ASSFTSVQYVKA MEKWKKW VGASLVALTLPF SVNGEAKA

Extr Cyt

4.37 4.82

41.11 21.07

MKK ACSLFVAVIVFL LFMPYSFASS MKK VFSTCLILFFLFL SKDAYA

a

The AIC gene numbering is according to the NCBI taxonomy database for strain B. lehensis G1. The annotation was primarily based on the genome annotation of B. leheniss G1. c The subcellular localization of proteins was predicted by using Gpos-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/Gpos-multi/); Extr: Extracellular; Cyt: Cytoplasm; V: Variety. d The probability of signal peptide was predicted for the proteins found in the secreted proteomes using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and subjected to Phobius (http://phobius.sbc.su.se/) for the functional domain grouping (n-, h- and c-region where h-region amino acids are shown in bold). Underlined amino acids in the GAP’s n-region are basic or aromatic residues. b

signal peptides for recombinant protein secretion in E. coli. To identify secreted proteins with signal peptide, the TCA-precipitated proteins collected from supernatant fraction were subjected to 2-DE analysis, producing an extracellular proteome map as shown in Fig. 1. From the map, 87 clearly identifiable protein spots by mass spectrometry were analyzed [30]. The identified proteins were grouped into 15 categories according to their functions, where they are mostly implicated in the metabolism of carbohydrates and related molecules (20%), cell wall (12%), metabolism of nucleotides and nucleic acids (11%) and proteins of unknown function (12%) [30]. Based on the 2-DE gel, protein spot with the greatest abundance was identified as flagellin, a polymeric protein that is the principal component of bacterial flagella, in agreement with the physiology of the Bacillus sp. secretome [31]. Besides that, a few proteins such as GlcNAc-binding protein A, CGTase and endopeptidase lytE were identified at more than one position on the 2-

[28,29]. A total of 1 ml of substrate buffer containing 4 mg/ml of orthonitrophenyl-β-galactoside (ONPG) in 0.1 M phosphate buffer (pH 7.4) was added with 0.1 ml of sample before it was incubated in 37 °C water bath for 10 min. The reaction was stopped by adding 0.5 ml of 1 M sodium carbonate and the absorbance was read at 420 nm. One unit of enzyme activity was defined as the amount of enzyme that forms 10−8 moles of ONPG per min under the experimental condition.

3. Results 3.1. Extracellular proteins and their signal peptides In this study, proteomic analysis of B. lehensis G1 on starch-containing medium was used for identification of highly secreted proteins. Naturally identified secreted proteins lead to finding of new potential 4

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glucanase 1 (BGL(4)) are the three clones exhibited improved excretion level of 735%, 305% and 176%, respectively. Meanwhile, a reduction in extracellular CGTase activity was observed for the rest of the transformants, where signal peptide of endonuclease/CDSuclease/phosphatase (ECDS) exhibited the lowest excretion CGTase activity (14% when compared to G1). Therefore, different signal peptides fused with CGTase can cause variation in expression, secretion and excretion level of the enzyme activity. Apparently, secretory production of recombinant proteins always correlate with cell lysis and has been seen as an obstacle for extracellular enzyme production in E. coli [34] even with the use of signal peptide. In this study, the occurrence of cell lysis was evaluated using βgalactosidase as an indicator [28,29]. Our results demonstrated that, after a 24 h cultivation period, signal peptide of endo-beta-1,3-glucanase (BGL) showed the highest value of β-galactosidase activity in the culture medium, 19.1 U/ml, whereas signal peptide of minor extracellular protease (MEP) exhibited the lowest value of 5.7 U/ml (Fig. 3). Among all transformants, BGL has a lower level of CGTase production with higher cell lysis, which indicates that BGL signal peptide is not suitable for extracellular production of CGTase in E. coli. Interestingly, GAP, which excretes the highest level of CGTase in the extracellular medium, has an increased of only ∼1.7 fold the amount of β-galactosidase released into the medium when compared to G1. To determine the quantitative relation between β-galactosidase activity and CGTase activity in the extracellular milieu of all the recombinant clones, the ratio of cell lysis per excretion was calculated (Fig. 4). The lower the ratio value, the lesser the cell lysis per excretion. Based on the results, GAP has the lowest ratio of cell lysis per excretion followed by 1853, which implies that it has high level of extracellular CGTase and low cell lysis. On the contrary, ECDS has the highest ratio value, which resulted from low yield of secreted protein with high cell lysis. Apart from the levels of recombinant proteins in different cell compartments, a signal peptide can also gives an impact to the secretion rate of the protein [35]. It is due to the many interactions involved between signal peptide and many proteins, chaperones and translocation machineries along the translocation process. To explore this aspect, the secretion rate of the fourteen recombinant clones was calculated (Fig. 5). Based on the results, ECDS has the lowest secretion rate of 2.0 U/ml/h which lead to low yield of CGTase in periplasm compartment (27% when compared to G1). Interestingly, the best performing signal peptide, GAP, displayed the highest secretion rate (45.6 U/ml/h) among all recombinant clones followed by 1853 and G1, which exhibited almost the same secretion rates, 28.5 U/ml/hr and 29.0 U/ml/ h, respectively. On the other hand, presence of signal peptide can also modulate protein stability and inclusion bodies formation [36]. SDS-PAGE was used to compare the insoluble fractions of E. coli harboring plasmids with heterologous signal peptides. As shown in Fig. 6, no inclusion bodies were observed in the insoluble fractions of CSP, signal peptide of chitinase (Chit) and signal peptide of trifunctional nucleotide phosphoesterase protein (TNPP), which may be due to low CGTase expression level in the cytoplasm (Fig. 3). Although GAP has the highest yield of secreted proteins in the extracellular milieu among all the recombinant clones, inclusion body formation was still detected on the SDS-PAGE. This indicates that GAP cells overproduced CGTase after a 24 h expression period where the enzyme concentration exceeds the solubility limit for the cytoplasm, thus precipitates from solution in the cytoplasm.

DE gel (Fig. 1). This could be due to post-translational modification, partial degradation or incomplete denaturation prior to electrophoresis, as reported in other studies [15,32]. Extracellular proteins are usually synthesized as proteins carrying cleavable signal peptides. Although extracellular proteins alone could be used as a fusion partner for recombinant protein secretion such as OsmY [15], there is limitation for high level secretion of targeted protein. Therefore, in this study, the presence of signal peptide in the identified proteins was predicted using the following criteria; (1): having D-score provided by the SignalP algorithm above the cut-off value for gram-positive bacteria (0.45) and (2) predicted by another signal peptide prediction tool (Phobius). Based on the analysis, fourteen signal peptides were further studied in E. coli expression system, as listed in Table 3. 3.2. Overexpression and Secretion Studies in E. coli The 14 signal peptides from B. lehensis were selected to assist in the secretion of the reporter protein (CGTase) across the cytoplasmic membrane of E. coli BL21(DE3). Overexpression and excretion studies were conducted using pET-21a plasmid as an expression vector under the IPTG-inducible T7 promoter. The efficiency of each signal peptides were tested on agar plate where the large halozone indicates efficient signal peptide in directing the proteins outside of the cell. As shown in Fig. 2, GAP exhibited larger halo zone when compared to other transformants, implies that secretion level of GAP was qualitatively higher than the rest of the transformants. As a negative control, W/O (recombinant clone without signal peptide) displayed no visible halo zone around the colony thus justifying that CGTase alone without signal peptide remained in the cytoplasm. The recombinant proteins expressed in E. coli can be directed to three different locations namely the cytoplasm, the periplasm or the extracellular environment. Since G1 is the native signal peptide used to direct CGTase in B. lehensis and has been widely used before [16,17,33], we compared and evaluated the applicability of heterologous signal peptides with G1 as the control. After 24 h induction at 30 °C, CGTase activities of all clones in all three compartments were measured (Fig. 3). Interestingly, GAP, signal peptide of cell surface protein (CSP) and signal peptide of hypothetical protein AIC94431 (1853) showed a 205%, 115% and 109% increase of periplasmic relative CGTase activity, respectively, as compared to G1. In addition, for CGTase extracellular secretion, GAP, 1853 and signal peptide of endo-1,3(4)-beta-

3.3. Secretion profile of GAP Fig. 2. Starch hydrolysis activity of transformants harboring the signal peptides-CGTase fusion genes. Halo zones were not observed from strains lacking the signal peptide (W/O). The formation of halo zones surrounding colonies was induced by growing the E. coli cells on LB-starch plates containing 1% (w/v) soluble starch, 0.5 mM lactose and 100 μg/ml ampicillin at 30 °C. Halo zones were revealed after treatment with iodine solution.

The best signal peptide (GAP) identified in secretome study was compared with G1 to further analyzed its efficiency, as illustrated in Fig. 7. The increasing pattern of secreted CGTase overtime was observed in the SDS-PAGE gel and western blot, conincided well with the 5

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Fig. 3. Effects of signal peptides on CGTase activities in the cytoplasm, periplasm and extracellular medium. Samples from the cytoplasm, periplasmic and extracellular fractions were taken at 24 h postinduction, respectively. Secretion activity of the transformants was calculated relative to native signal peptide (G1) activity set to 100%.

periplasmic space in the first hour after induction, approximately 5.9fold increase compared to G1 (3.0 U/ml). Subsequently, the CGTase activity in periplasm of GAP increased and maintained until 4 h postinduction (166.5 U/ml), when no further increase of CGTase was detected. Moreover, at 2 h post-induction, GAP excreted the CGTase to the culture medium and steadily increased until it reached a plateau at 8 h post-induction and eventually reached 637.5 U/ml enzyme activities at 24 post-induction.

3.4. In silico analysis of signal peptides The amino acid sequences of signal peptides were subjected to Phobius webserver analysis for the functional domain grouping (n-, hand c-region), as listed in Table 3. Additionally, different physicochemical characteristics of signal peptides were analyzed using ProtParam tool (Table 4). The length range of all signal peptides was between 22 and 34 amino acids. The n-region net charge was −1 for G1, +1 for CSP and 1853, +2 for signal peptide of endopeptidase lytE (LytE), BGL, TNPP, signal peptide of hypothetical protein AIC96089 (3542) and signal peptide of hypothetical protein AIC96118 (3571) and +3 for the other six signal peptides. The overall hydrophobicity of the signal peptides were compared using GRAVY score, which is calculated as the sum of hydropathy values of all the amino acids, divided by the number of residues in the sequence. Based on the analysis, CSP has the highest hydrophobicity value of 1.21 while TNPP exhibited the lowest, 0.122. SignalP used an algorithm to predict the probability of an amino acid sequence functioning as a signal peptide, known as the D-score [37]. Higher D-scores indicates that the corresponding sequence has a higher chance of functioning as a signal peptide, and vice versa. Our results showed that there seemed to be no correlation between high excretion level and the calculated D-scores. The screening revealed that signal peptides with D-scores above 0.7 such as signal peptide of hypothetical protein AIC94426 (1848), ECDS, LytE, MEP and 3542 showing reduction in CGTase excretion level compared to G1, as summarized in Table 4. Based on the results, we can conclude that CGTase secretion in E. coli preferred signal peptide with the combination of a positively charge n-region, a lower hydrophobicity (0.5–0.7) and a Dscore between 0.5 and 0.7. Besides, one of the common causes for failures in recombinant gene expression is related to imbalance in codon usage. Rare codons tend to slow down the translation rates and accuracy, and thus influence protein translocation. The presence of rare codons in 14 signal peptide’s nucleotide sequences was calculated (Table 4). Based on the

Fig. 4. Ratio of cell lysis per excretion of recombinant clones. The ratio was calculated as β-galactosidase/CGTase unit activity detected in the medium.

Fig. 5. Secretion rate of recombinant clones. Rate of secretion was calculated as Unit activity in periplasmic at 4 h/4 h.

increasing of CGTase enzyme activity for GAP. At the end of the cultivation period (24 h post-induction), GAP showed increases in CGTase excretion up to ∼5-fold (637.4 U/ml) compared to G1 (119.6 U/ml). As shown in Fig. 7B, GAP mediated the CGTase (17.8 U/ml) into the 6

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Fig. 6. Solubility comparison of recombinant CGTase in the insoluble fraction of 14 transformants in this study, at 24 h post-induction. Lane M, protein marker (10–250 kDa, NEB).

induction compared to modified G1 signal peptide (M5) reported in a previous study [17]. High secretion rate of GAP allows large amounts of synthesized pre-CGTase to be translocated through the inner membrane in a timely manner, then to the periplasm, thereby keeping the preprotein translocation channel open. This explains why a higher amount of CGTase was obtained in GAP extracellular medium (Fig. 7B). High rate of secretion usually correlates with regions of signal peptide and their amino acid composition, as signal peptides are required in at least three stages: (a) keeping the pre-proteins translocation competent [39]; (b) interaction with other components of the secretion machinery [40] and (c) as a topological determinant for preproteins in the membrane [41]. A typical signal peptide exhibits a similar tripartite structural organization: a positive charged n-region, a helical hydrophobic h-region and a slightly polar c-region containing signal peptidase cleavage site [42]. In this study, increased n-region’s net charge of signal peptide from −1 (G1) to +3 (GAP and BGL(4)), +1 (CSP and

calculation, G1, GAP, TNPP and 3571 showed no rare codons usage while Chit exhibited five rare codons, which may cause a problem during protein translation.

4. Discussion Extracellular secretion in E. coli often encounters problems such as low secretion and cell lysis. One of the most pivotal factors that has a crucial influence on all stages of secretion pathway and ultimately, on the amount of the secreted protein is the signal peptide [38]. The aim of this study was to screen signal peptides of naturally-secreted proteins of B. lehensis G1 which can improve the secretion of recombinant proteins in E. coli. A close inspection of the 14 signal peptides upon overexpression suggests that GAP is the optimal signal peptide with high secretion rate (Fig. 5). The excretion yield of CGTase to the extracellular medium by GAP increased to ∼2.9-fold higher at 12 h post-

Fig. 7. Secretion profiles. The culture was grown at 37 °C until OD600 nm reached 0.7, and it was then induced with 0.01 mM IPTG and incubated at 30 °C for another 24 h. All experiments were conducted under identical conditions and performed in triplicates. CGTase activity is depicted in the extracellular medium (filled circles) and periplasm (open circles). Beta-galactosidase activity detected in the culture medium (grey bar) was used as an indicator of cell lysis. SDS-PAGE analysis of recombinant CGTase in the extracellular medium at 0 h till 24 h post-induction that were taken from recombinant E. coli, G1 (C) and GAP (D), grown in LB medium. Western blot analysis of recombinant CGTase in the extracellular medium that were taken from G1 (E) and GAP (F), corresponding to the SDS-PAGE analysis. Lane M, protein marker (10–250 kDa, NEB). 0, 0hr; 1, 1hr; 2, 2hr; 3, 3hr; 4, 4hr; 6, 6hr; 8, 8hr; 10, 10hr; 12, 12hr; 24, 24hr.

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peptide by calculating the D-score. High D-scores calculated by SignalP do not concurrently indicate high translocation efficiencies of the secreted protein nor do low D-scores around 0.5 indicate low yield of enzyme secretion. Our findings agree with the notion that the calculated D-scores with the SignalP prediction tool did not reveal any significant similarities with recombinant protein secretion level, as reported by Degering et al. [52] and Brockmeier et al. [12]. Another aspect of this work relates to the fact that codon bias influenced not only the translation steps but also protein production and protein folding [53], and thus eventually affected the secretion rate of the desired protein. In this study, Chit composed of the highest percentage of rare codon usage in the sequence (Table 4), resulted in no inclusion bodies formation (Fig. 6) with significantly reduced cytoplasmic CGTase activities, compared to G1 (Fig. 3). This implies that rare codon usage slow down the translation rate across the signal peptide caused low protein yield. Based on the obtained results, GAP has improved secretion level as rare codon was not predicted in the signal peptide sequence. In contrast, optimized codons in the signal sequence of maltose binding protein caused significant reduction in secretion level [54]. Recently, proteome-based approach has been reported for the identification of fusion partners (secreted proteins and their signal peptide) to improve secretion in E. coli [15]. The authors describe that the use of secreted proteins with enterokinase can resulted in extracellular production of recombinant proteins. There are several limiting factors when using a carrier protein to secrete desired protein includes the need to cleave fusions and the size of the fusion partner as large proteins are more burdensome to be secreted [6]. However, we show in this study, instead of the whole mature secreted protein with its signal peptide, the heterologous signal peptide from Bacillus sp. is enough for successful secretion of the recombinant protein in E. coli. The secretion level of recombinant protein can be improved by finding the right balance through optimal signal peptide, since heterologous signal peptides used in this work were interestingly resulted in an improved secretion level. In summary, we are able to identify naturally secreted proteins in B. lehensis G1 by proteome analysis. Among the tested signal peptides, GAP was found to excrete desired enzyme into the medium at significantly higher level than others upon over-expression. In the signal peptide research, GAP could be a good candidate for further experimental analysis of recombinant proteins secretion in the future. Furthermore, the strategy of employing the signal peptides from Bacillus sp. is useful for the excretory production of recombinant proteins using the well-established E. coli system.

Table 4 In silico analysis of signal peptides. Signal peptide

Length (aaa)

Net charge of nregion b

Hydrophobicityc

D-scored

Rare codonse

Relative CGTase activity in culture medium (%)

G1 GAP 1853 BGL(4) 1848 3571 3542 MEP BGL Chit LytE CSP TNPP ECDS

29 34 26 25 25 22 25 35 24 27 29 29 27 29

−1 +3 +1 +3 +3 +2 +2 +3 +2 +3 +2 +1 +2 +3

1.090 0.685 0.504 0.640 1.096 1.105 1.500 0.546 1.050 0.874 0.807 1.210 0.122 0.734

0.660 0.489 0.698 0.592 0.812 0.606 0.717 0.718 0.649 0.627 0.735 0.688 0.545 0.741

0 0 2 1 1 0 1 2 3 5 1 2 0 1

100% 735% 305% 177% 97% 92% 72% 64% 56% 50% 47% 44% 26% 14%

a

aa, amino acids. The net charge of the N-region was calculated with amino acids aspirate and glutamate defined as −1, arginine and lysine defined as +1 and any other amino acid defined as 0. c ProtParam tool (http://web.expasy.org/protparam/) was used to calculate total hydrophobicity of signal peptide based on Grand average of hydropathicity (GRAVY). d D-score calculated by SignalP 4.1(http://www.cbs.dtu.dk/services/SignalP/). e Nucleotide sequences were analyzed using rare codon calculator (RaCC) at http:// nihserver.mbi.ucla.edu/RACC/. b

1853) yielded a higher level of CGTase secretion compared to G1 (Fig. 3). Previous study reported that interaction of signal peptide with SecA (pre-protein translocase) increases with the number of positive charges in the n-region [43]. Additional arginine (basic) residues in signal peptide that lead to increased n-region’s charge notably improved the secretion levels of human lysozyme [44]. However, it should be noted that the presence of one or more basic amino acids in the nregion is probably essential but not a must for a signal peptide. Our results suggest that increased n-region’s positive charge does not necessarily guarantee a good level of secreted enzyme activity, such as exhibited by ECDS and MEP (Fig. 3). Similarly, previous observation by Morioka-Fujimoto et al. [45] confirms that an increase in n-region’s positive charge of signal peptide had no effect on the secretion of recombinant protein. Though the positive charge of the n-region is essential for recognition by pre-protein translocase (SecA) to initiate protein translocation and also to interact with the negatively charged lipids at the cell membrane [46,47], combination of the signal peptide, the desired protein and the host strain is still to be considered. Signal peptides with highly hydrophobic regions can drive a rapid rate of transport in the presence of a negatively charged n-region [48]. For that reason, signal peptide hydrophobicity is one of the decisive factors for signal peptide’s affinity towards signal recognition particle (SRP) and the rate of protein secretion [49,50]. Increasing the hydrophobicity of the h-region can improve secretion and production of proteins in E. coli [7]. In contrast, the three best-performing signal peptides (GAP, 1853 and BGL(4)) have a lower hydrophobicity compared to G1 (Table 4), indicates that stronger binding affinity of SRP and signal peptide does not necessarily improve CGTase secretion. A similar observation [17] revealed that a decrease in hydrophobicity due to addition of a G-turn leads to increased secretion of recombinant CGTase into the periplasmic space. Basic n-region and hydrophobic hregion of a signal peptide are closely interrelated, and may act as a unit, where an ideal unit can provide optimal secretion of the desired protein [51]. In the present study, SignalP prediction tool was used to predict the probability of a particular sequence if it could functions as a signal

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