Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli

Journal of Biotechnology 135 (2008) 319–325

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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli Yin Xu, Frances C. Foong ∗ School of Biotechnology & Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia

a r t i c l e

i n f o

Article history: Received 11 February 2008 Received in revised form 23 April 2008 Accepted 2 May 2008 Keywords: Cellulose binding domain EngD Solubility Immobilization Protein fusion Purification Amorphous cellulose Affinity tag

a b s t r a c t Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PTlinker, PTCBDEngD , cellulose binding domain of C. cellulovorans cellulose binding protein A, CBDCbpA , cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBDEngD . Insoluble domain Coh6 was solubilized when fused with PTCBDEngD . In contrast, fusion with CBDCbpA resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBDEngD and Cip7PTCBDEngD were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBDEngD eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBDEngD can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Escherichia coli is an important host cell for recombinant protein expression because of its easy manipulation and high biomassto-cost ratio. Some heterologous proteins form inclusion bodies during hyperexpression in E. coli and one reason is the nascent proteins do not have enough time to fold correctly. Inclusion bodies can be solubilized in vitro with the process of denaturation and refolding. However, protein yield is reduced and protein bioactivity is not guaranteed. In addition, incorrectly folded proteins are difficult to separate from correctly folded proteins. Another strategy to increase the proportion of bioactive proteins is to decrease the formation of inclusion bodies in vivo. This can be accomplished by a change in expression temperatures (Yeh et al., 2005), reduced medium nutrition and inducer concentration, and/or fusing with a domain which can increase the solubility of the chimera (Kapust and Waugh, 1999). Commonly used fusion partners for E. coli include Salmonella japonicum glutathione S-transferase (GST) (Smith and Johnson, 1988), E. coli maltose-binding protein (MBP) (Guan et al., 1988), thioredoxin (LaVallie et al., 1993), NusA, grpE

∗ Corresponding author. Tel.: +61 2 93853872. E-mail address: [email protected] (F.C. Foong). 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.05.004

and bacterioferritin (BFR) (Davis et al., 1999). Human protein disulfide isomerase (PDI) (Liu et al., 2006), bacteriophage T7 protein kinase (T7PK) and E. coli chaperone Skp (Chatterjee and Esposito, 2006) have also been recently reported. Cellulose binding domains (CBDs) are non-catalytic domains found mostly in cellulolytic microorganisms, either as a domain on the scaffolding protein of the multi-enzyme complex cellulosome, e.g. CBD from Clostridium cellulovorans scaffoldin CbpA (CBDcbpA ), or as a domain of the non-cellulosomal cellulase, e.g. CBD from Cellulomonas fimi endoglucanase CenA (CBDCenA ) and exoglucanase Cex (CBDCex ). Cellulosomal cellulase such as EngB of C. cellulovorans do not have a CBD but instead have a duplicated sequence which is recognized as dockerin for interaction with the complementary cohesin domain on the scaffolding protein (Foong and Doi, 1992) of cellulosomes. Cohesins are another category of non-catalytic domains in cellulosomes. They are located on the scaffolding protein and can interact with the dockerin domains. Cohesin–dockerin interaction is one of the most tenacious protein–protein association; however, the formation and stabilization of cohesin–dockerin complexes is Ca2+ dependent so the complex can be disrupted by chelating reagents such as EDTA (Choi and Ljungdahl, 1996). In nature, CBDs enhance enzyme activities by concentrating the enzyme on the substrate surface and/or are involved in the

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disruption of non-covalent interaction between adjacent substrate molecules (Din et al., 1991). In research, CBDs are used as affinity tags for purification or immobilization. CBDCenA was fused with alkaline phosphatase (PhoA) with a proline-threonine rich region (PT-linker) and an IEGR sequence recognized by Factor Xa for cleavage when the chimera was expressed in E. coli. CBDPT-PhoA was purified on the cellulose CF1 (Sigma) column and the PhoA domain was cleaved from the CBD domain by Factor Xa digestion. The column could be reused for up to 10 cycles without a decrease in efficiency (Greenwood et al., 1994). Fusion protein containing CBDcbpA and an antibody binding domain protein LG was coated on cellulose hollow fibre. The CBDcbpA -LG coated fibre efficiently immobilized CD34+ cells labelled with mouse IgG2a monoclonal antibody MHCD3400 (Craig et al., 2007). CBDcbpA has been well characterized and commercialized as a fusion domain for protein purification using cellulose matrix (Novagen). This CBD was found to have good affinity for highly crystalline cellulose such as cotton, cellulon and Avicel PH101. It also has affinity for chitin. But CBDcbpA ’s binding affinity for regenerated amorphous cellulose such as fibrous and microgranular cellulose was much lower. In addition, CBDcbpA and some CBDcbpA fusion proteins were insoluble when expressed in E. coli (Goldstein et al., 1993). In many circumstances, cellulose is regenerated to different forms such as membranes, fibres and microgranules. During the regeneration process, the crystallinity of the cellulose is reduced. These regenerated cellulosic materials can be used for making bioreactors and filling purification columns. In this study, effort was directed to looking for CBDs which can fulfill two requirements: high affinity for regenerated cellulose as well as an increase in solubility of the recombinant proteins when expressed in E. coli. One of the potential candidates is a putative CBD from C. cellulovorans endoglucanase-xylanase EngD. EngD is believed to possess a catalytic domain at the N-terminus, a cellulose binding domain (CBDengD ) at the C-terminus, and a PT-linker connecting the two domains. Western blot analysis showed that native EngD and recombinant protein containing CBDengD could bind Avicel (Foong and Doi, 1992). In domain swapping experiments with E. coli as host for expression of recombinant proteins, the putative CBD from EngD was found to have a solubilization effect on the catalytic domains of the cellulosomal enzyme, EngB (Murashima et al., 2003). The PT-linker was also found to influence the solubility and catalytic activities of EngD proteins (Yeh et al., 2005). However, CBDengD ’s affinity for crystalline or regenerated cellulose has never been characterized or quantified. In this study, we constructed different fusion proteins composed of PT-CBDengD or CBDcbpA , cohesins from various Clostridium species or LG, a recombinant antibody binding protein, to investigate the binding specificities of CBD fusion proteins, to compare the effect of different CBDs on fusion protein solubilities and to screen appropriate fusion proteins in terms of their solubility and purification characteristics for future use in developing interactive cell immobilization systems.

Table 1 Names and sequences of PCR primers used for insert preparation Primer name

Primer sequence (5 to 3 )

Fcbd-EcoRI Rcbd-SalI Fpt-cbd-EcoRI

ACA CCT ACG AAT TCA CAA TCA GC TG CTC GTC GAC TTT TAC TGT GC TA AAG AAT TCT GGT GGG TCA AG

Fcip7-SalI Rcip7-XhoI Fcip7-BamHI Rcip7-EcoRI

CAT CAC GTC GAC GAT CTG GAT G CG GCG GCT CTC GAG ATC TC CAT CAG GAT CCG GAT CTG GAT G GTG GCG GGA ATT CAA GAC ATC TC

Fpt-cbd-SalI Rpt-cbd-SalI

GC AAG CTT GTC GAC TTT TAC TGT GC AT CCG GTC GAC GGT GGG TC

Fcoh6-BamHI Rcoh6-BamHI FcipC1-BamHI RcipC1-BamHI

T CGG GAT CCG AAT TCG AGC TCC G GTG GTG CGG ATC CCA AAC TGT AGC TG GGT CGG GAT CCG AAT TC G GTG CTC GGG ATC CGC TAC

Fcip7-EcoRI

CG ATA TCG GAT CCG AAT TCG ATT GAT CTG GG

Rcip7-NotI

GC GGC CGC AAG TTG GCT TGT CGA C

Highlighted in bold are restriction enzyme sites and underlining indicate altered bases.

Plasmids pET23-lgdoc, pET34-Cip7 and pET23-LG (Craig et al., 2006), pET23-engD (Yeh et al., 2005), pET23-coh6 and pET23-cipC1 were constructed in previous studies. 2.2. Construction of pET vectors Gene fragments were PCR amplified with primers and introduced restriction sites (Table 1) and TA cloned into pGEM-T Easy. Inserts were sequenced in both directions to confirm the fidelity before restriction digestion with the appropriate enzymes and subcloned into pET vectors. 2.2.1. Construction of pET23-cbdengD and pET23-pt-cbdengD cbdengD and pt-cbdengD were PCR amplified from pET23-engD using forward primers Fcbd-EcoRI and Fpt-cbd-EcoRI, respectively and the same reverse primer Rcbd-SalI. The fragments were digested using EcoRI and SalI and subcloned into pET23b+. The final constructs were named pET23-cbdengD and pET23-pt-cbdengD . They were used for the expression of CBDengD and PT-CBDengD with a fused C-terminal His-tag. 2.2.2. Construction of pET23-cip7-cbdengD and pET23-cip7-pt-cbdengD The cip7 domain was PCR amplified from pCip7 using primer set Fcip7-BamHI/Rcip7-EcoRI. The cip7 fragment was digested using BamHI and EcoRI and subcloned into pET23-cbdengD and pET23pt-cbdengD , respectively. The final constructs were named pET23cip7-cbdengD and pET23-cip7-pt-cbdengD , in which cip7 was located at the N-termini of cbdengD and pt-cbdengD . His-tags were at the C-termini of these fusions.

2. Materials and methods 2.1. Bacterial strains and vectors E. coli strain XL10 Gold (Stratagene, La Jolla, CA, USA) and pGEMT Easy (Promega, Madison, WI, USA) were used for cloning work. E. coli strain BL21(DE3)pLysS (Promega) and vectors pET23b+ and pET34b+ (Novagen, USA) were used for protein expression. E. coli strain M15(pREP4) with pCip7 (Kataeva et al., 1997) was kindly provided by Dr. Pierre Beguin (Institute Pasteur, Paris, France) and used for both cloning and expression of cip7.

2.2.3. Construction of pET23-cbdengD -cip7 and pET23-pt-cbdengD -cip7 The cip7 domain was PCR amplified from pCip7 using primer set Fcip7-SalI/Rcip7-XhoI. The cip7 fragment was digested using SalI and XhoI and subcloned into pET23-cbdengD and pET23-pt-cbdengD , respectively. The final constructs were named pET23-cbdengD -cip7 and pET23-pt-cbdengD -cip7, in which cip7 was located at the Ctermini of cbdengD and pt-cbdengD . His-tags were at the C-termini of these fusions.

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2.2.4. Construction of pET23-lg-pt-cbdengD Fragment pt-cbdengD was amplified from pET23-pt-cbdengD with primer set Fpt-cbd-SalI/Rpt-cbd-SalI. The pt-cbdengD fragment was digested with SalI and subcloned into pET23-lg. As the fragment had identical sticky ends generated by single digestion, it could be inserted into pET23-lg in opposite directions, resulting in different restriction patterns. HindIII was used to determine the direction of the inserts and to screen for the correct clone, pET23-lg-pt-cbdengD . 2.2.5. Construction of pET23-coh6-pt-cbdengD and pET23-cipC1-pt-cbdengD Fragments coh6 and cipC1 were amplified from pET23-coh6 and pET23-cipC1 using primer sets Fcoh6-BamHI/Rcoh6-BamHI and FcipC1-BamHI/RcipC1-BamHI. The fragments were digested with BamHI and subcloned into pET23-pt-cbdengD . SalI digestion was used to determine the direction of the inserts and to screen for the correct clones, pET23-coh6-pt-cbdengD and pET23-cipC1-ptcbdengD . 2.2.6. Construction of pET34-coh6 and pET34-cipC1 Fragments coh6 and cipC1 were digested from pET23-coh6 and pET23-cipC1 by EcoRI and XhoI and cloned into pET34b+. The final plasmids were named pET34-coh6 and pET34-cipC1. 2.2.7. Reconstruction of pET34-Cip7 The original version of pET34-Cip7 (Craig et al., 2006) had an amber stop codon between cip7 and the His-tag. It was expressed as an insoluble protein in E. coli JM109, which had an amber suppressor, SupE44 to read through the amber stop codon and fusion with the His-tag. Primer set Fcip7-EcoRI/Rcip7-NotI was designed to replace the amber stop codon with a sense codon so that the newly constructed pET34-cip7 could be expressed in E. coli strain BL21. 2.3. Protein expression and lysate samples preparation pCip7 was expressed in M15 (pREP4). Other genes or fusion genes constructed in pET vectors were expressed in BL21(DE3)pLysS. Cells were induced with 0.4 mM IPTG when culture OD600 reached 0.4–0.5 and cultured for a further 3 h in LB (Luria-Bertani) broth with antibiotics (50 ␮g/mL Ampicillin or 25 ␮g/mL Kanamycin) at 30 ◦ C and 200 rpm shaking. The cells were then pelleted by centrifugation at 10,000 × g for 6 min and resuspended in 1/50 culture volume of 20 mM bis–tris buffer (pH6.0). Cells were sonicated with Ultrasonic Cell Disruptor Sonifier® B-30 Model 200 (Branson, USA) at strength setting of 6.5 with the cycle of 45 s burst followed by 15 s cooling for 5 cycles; or cells were disrupted by high pressure homogenization with APV Homogeniser Motor type 7.30 VH (Albertslund, Denmark). The cell debris (insoluble fraction) was pelleted by centrifugation at 10,000 × g for 1.5 h and the supernatant contained soluble proteins (soluble fraction). 2.4. Protein purification All purification was performed on an FPLC system (Pharmacia) fitted with automated computer controller giving continuous readings of conductivity and absorbance at 280 nm. Only the soluble fractions were used for purification. Cip7-PTCBDengD , CBDcbpA -Cip7, LG-Doc and LG were purified by immobilized metal affinity chromatography (IMAC) on a 5 mL prepacked HisTrapTM HP column (Amersham Biosciences). The Ni2+ charged column was equilibrated with IMAC binding buffer (20 mM NaH2 PO4 , pH 7.4, 0.5 M NaCl, 20 mM imidazole). Protein lysate was then applied to the column followed by washing with IMAC binding buffer. Proteins were eluted with IMAC elution buffer (IMAC binding buffer with 500 mM

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imidazole) and fractionated using an imidazole step. LG-PTCBDengD was purified by anion exchange chromatography (AEC) on the XK 26/20 column (Pharmacia) filled with 60 mL Q SepharoseTM Fast Flow (Amersham Biosciences). The column was equilibrated with 20 mM bis–tris (pH 6.0) before loading soluble protein extract. Protein was eluted with 20 mM bis–tris (pH 6.0) plus 500 mM NaCl and fractionated using a NaCl gradient. CBDcbpA -LG was purified by AEC followed by IMAC. Purified proteins were dialyzed against HBS buffer (20 mM HEPES pH 7.4, 150 mM NaCl). Purified proteins were run on NuPAGE gels and documented by photographing. Densitometry analysis was done by the program ImageJ (NIH website). The relative amount of proteins was determined by plotting the lanes to peak graphs according to the intensity and shape of the bands. The purity of target protein is the ratio of the area under the peak for the target protein to the area under the peaks for total proteins: Purity(%) =

Target peak area × 100 . Total peak area

2.5. Comparison of solubilities Equilibrated lysate, soluble and insoluble fractions were prepared for each cell lysate sample and ran on NuPAGE gels and analyzed by densitometry as described. Solubility of the target protein is the ratio of the area under the peak for the soluble target protein to the sum of area under the peaks for soluble and insoluble target protein: Solubility(%) =

Intensity of soluble protein × 100 Sum of intensity of soluble and insoluble protein

Solubilities of CBDengD , PTCBDengD , Cip7-CBDengD , Cip7-PTCBDengD , CBDengD -Cip7 and PTCBDengD -Cip7 at the third hour after cell induction were compared. Solubilities of single domains (X) and their CBD chimeras (CBDcbpA -X and X-PTCBDengD ) at the third hour after induction were also compared. 2.6. Cellulose binding assays Specific binding of recombinant proteins to regenerated amorphous cellulose, Cuprophan fibre (Membrana GmbH, Wuppertal, Germany) was assessed. Cuprophan fibre was snap frozen in a small quantity of liquid nitrogen and ground to a fine powder by mortar and pestle. Cuprophan binding assays were performed in 1.5 mL microcentrifuge tubes and fractions in each step were recovered as flow through by centrifugation. Soluble cell lysate (0.5 mL) of different concentrations was mixed thoroughly with 0.1 g of ground Cuprophan in an eppendorf tube. The mixture was incubated at 4 ◦ C for 2 h in the binding step followed by washing Cuprophan with equal l.v. (loading volume) of wash buffer (20 mM HEPES buffer, pH 7.5, 1 M NaCl, 0.05%, v/v Tween-20) 3 times. In the elution step, the fibre was transferred to a fresh microcentrifuge tube and the Cuprophan-bound proteins were eluted in equal l.v. of 1× SDS loading dye. Proteins in each fraction were visualized on 4–12% NuPAGE gels stained with Coomassie dye. 3. Results and discussion 3.1. Comparison of solubilities of CBDcbpA -X chimeras and X-PTCBDengD chimeras CBDengD (14.6 kDa), PTCBDengD (18.0 kDa), Cip7-CBDengD (30.6 kDa), Cip7-PTCBDengD (34.0 kDa), CBDengD -Cip7 (29.9 kDa) and PTCBDengD -Cip7 (33.3 kDa) were expressed in BL21(DE3)pLysS at 30 ◦ C, induced with 0.4 mM IPTG with an extended expression

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Table 2 Solubilities of CBDengD , PTCBDengD , Cip7-CBDengD , Cip7-PTCBDengD , CBDengD -Cip7 and PTCBDengD -Cip7 at different time points after IPTG induction Solubility (%)

2nd hour

3rd hour

4th hour

CBDengD PTCBDengD Cip7-CBDengD Cip7-PTCBDengD CBDengD -Cip7 PTCBDengD -Cip7

52 73 88 93 67 80

41 63 85 93 70 77

42 65 84 90 66 68

time of 4 h (gel photos not shown). Estimated solubilities for each protein were calculated and summarized in Table 2. As can be seen from the results, the solubilities of proteins containing a PT linker were slightly higher than the corresponding protein domains without the linker. The solubilities of CBDengD and PTCBDengD were further increased when fused to a Cip7 domain. Chimeric proteins with an N-terminal Cip7 have higher solubilities than those with a C-terminal Cip7. PTCBDengD was chosen for fusion with protein Cip7, Coh6, CipC1 and LG at their C terminus and the solubility of these chimeras were compared with that of their counterparts containing an N-terminal CBDcbpA . Solubilities of protein Cip7, Coh6, CipC1 and LG, as well as their chimeric proteins fused with an N-terminal CBDcbpA or a C-terminal PTCBDengD were calculated and summarized in Table 3. As seen in Table 3, when fused with an N-terminal CBDcbpA , the solubilities of CBDcbpA -LG and CBDcbpA -Cip7 were comparable to that of their single domains, LG and Cip7, at over 80%. It was interesting to note that Coh6 which by itself was very insoluble but when fused with CBDcbpA at the N-terminus, solubility increased slightly from 19.6 ± 1.7% to 37.8 ± 6.3%; however, the solubility of CBDcbpA -CipC1 (18.8 ± 3.1%) was greatly decreased compared to CipC1 by itself (86.3 ± 4.7%). A C-terminal PTCBDengD did not introduce significant changes in solubilities of already highly soluble domains LG, Cip7 or CipC1 but was able to increase the solubility of insoluble Coh6 by three folds, from 19.6% to 59.8%. SDS-PAGE patterns of soluble and insoluble fractions of cell lysates from pET23-coh6, pET34-coh6, pET23-coh6-ptcbdengD and pET23-cipC1, pET34-cipC1 and pET23cipC1-ptcbdengD is also shown in Fig. 1. A similar phenomenon was found in a previous study on C. cellulovorans Endoglucanase EngB, when CBDengD was found to solubilize EngB-CBDengD fusions (Murashima et al., 2003). The mechanism of how a C-terminal PTCBDengD increases the solubility of fusion proteins is not clear. Kapust and his colleagues proposed a model for explaining how a fusion protein, MBP helps to solubilize an insoluble protein domain when it is located at the N-terminus of the fusion protein (Kapust and Waugh, 1999). We propose that the C-terminal-PTCBDengD Table 3 Estimated solubilities of X domains, X-PTCBDengD chimeras and CBDcbpA -X chimeras at the 3rd hour of expression Domain and chimera

Solubility (%)

Cip7 CBDcbpA -Cip7 Cip7-PTCBDengD

86.1 ± 2.8, n = 2 86.0 ± 1.4, n = 3 89.5 ± 4.9, n = 2

Coh6 CBDcbpA -Coh6 Coh6-PTCBDengD

19.6 ± 1.7, n = 3 37.8 ± 6.3, n = 3 59.8 ± 6.2, n = 3

CipC1 CBDcbpA -CipC1 CipC1-PTCBDengD

86.3 ± 4.7, n = 3 18.8 ± 3.1, n = 3 90.1 ± 3.1, n = 3

LG CBDcbpA -LG LG-PTCBDengD

88.2 ± 2.9, n = 2 84.8 ± 12.0, n = 2 89.0 ± 4.2, n = 2

“n” indicates the number of experiment repeats.

Fig. 1. Solubility analyses of Coh6, CBDcbpA -Coh6, Coh6-PTCBDengD , CipC1, CBDcbpA CipC1 and CipC1-PTCBDengD . Lane 1: Coh6 soluble portion; lane 2: Coh6 insoluble; lane 3: CBDcbpA -Coh6 soluble; lane 4: CBDcbpA -Coh6 insoluble; lane 5: Coh6PTCBDengD soluble; lane 6: Coh6-PTCBDengD insoluble; lane 7: marker; lane 8: CipC1 soluble; lane 9: CipC1 insoluble; lane 10: CBDcbpA -CipC1 soluble; lane 11: CBDcbpA CipC1 insoluble; lane 12: CipC1-PTCBDengD soluble; lane 13: CipC1-PTCBDengD insoluble. Arrows indicate the target protein bands.

increase the solubility of the fusion protein, Coh6-PTCBDengD through a similar mechanism, where CBDengD ’s sequestration of unfolded conformations prior to complete folding maintains the low level of unfolded Coh6 and frequency of self-association. CBDengD is thought to fold in a two-state kinetics manner so the fusion can reach equilibrium rapidly (Murashima et al., 2003). This feature is also thought to contribute to the expression of soluble CBDengD . Results in this study showed that the PT linker can also increase the solubility of chimeric proteins, in particular when it is between a cohesin and the CBDengD . It was shown in a previous study that the chimeric protein with the PT linker of EngD had the highest estimated percentage of soluble protein of different chimeric proteins (Yeh et al., 2005). NMR spectroscopic analysis on Cellulomonas fimi CBDcex showed that the linker is a flexible tether, without any predominant structure, between an independently folded catalytic domain and the binding domain (Poon et al., 2007). In prokaryotic cells, peptide folding follows directly the process of coupled transcription-translation therefore the structurally flexible PT linker may provide for the capability and extra time required by its fusion domains to fold correctly and independently. In addition, choosing a proper vector/host strain combination is an important factor which affects recombinant protein solubility. It was shown in a previous study that when pET34-Cip7 was expressed in JM109(DE3) or as vector pQE40-CBDCip7 in XL10 Gold, fusion protein CBDcbpA -Cip7 was expressed with a high proportion of inclusion bodies (Craig et al., 2006). When pET34-Cip7 was reconstructed to remove the amber stop codon and expressed in BL21(DE3)pLysS, the solubility achieved 86.0 ± 1.4%. 3.2. Protein purification 3.2.1. Purification of Cip7-PTCBDengD Soluble Cip7-PTCBDengD was purified by IMAC on a 5 mL Ni2+ charged HisTrapTM HP column with a starting purity of 20.9 ± 4.7% (n = 8). This protein started to be eluted with 60 mM imidazole and the peak was at 150 mM imidazole. Little protein was obtained at higher imidazole eluate. Cip7-PTCBDengD was in the form of a monomer (Fig. 2). The average purity of Cip7-PTCBDengD after IMAC was 92.0 ± 0.7% (n = 3).

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Fig. 4. Purification of CBDcbpA -Cip7 from cell lysate using IMAC. Lane 1: total protein from cell lysate; lane 2: flow through; lane 3: wash; lane 4: marker; lane 5: eluate at 50 mM imidazole; lane 6: eluate at 75 mM imidazole; lane 7: eluate at 150 mM imidazole; lanes 8 and 9: eluate at 300 mM imidazole; lanes 10 and 11: eluate at 500 mM imidazole. The arrows indicate the protein and size of CBDcbpA -Cip7 monomer. Fig. 2. Purification of Cip7-PTCBDengD from cell lysate using IMAC. Lane 1: total soluble protein from cell lysate; lane 2: flow through; lane 3: wash; lane 4: marker; lane 5: eluate at 50 mM imidazole; lane 6: eluate at 60 mM imidazole; lane 7: eluate at 150 mM imidazole; lane 8: eluate at 500 mM imidazole. Arrows indicate the protein and size of Cip7-PTCBDengD .

3.2.2. Purification of LG-PTCBDengD Soluble LG-PTCBDengD accounted for 26.5 ± 3.0% (n = 2) of total soluble proteins. Trials were performed to purify LG-PTCBDengD by IMAC and AEC. It was found that IMAC was not suitable for LG-PTCBDengD purification because it flowed through the Histrap column with other proteins at low imidazole concentrations (gel photo not shown). However, in AEC, little LG-PTCBDengD was present in flow through and wash fraction. Majority of purified LG-PTCBDengD was collected between 13.5 mS and 16.5 mS and in monomer form (Fig. 3). Average purity of LG-PTCBDengD after AEC was 88.2 ± 3.1% (n = 2). 3.2.3. Purification of CBDcbpA -Cip7 CBDcbpA -Cip7 was purified as Cip7-PTCBDengD at a higher starting purity of 37.0 ± 3.7% (n = 7). It was eluted at 150–500 mM

imidazole. The purity of the eluate decreased with the increase in imidazole concentration, from more than 95% at 150 mM to less than 85% at 500 mM. Most of the CBDcbpA -Cip7 was in the form of monomers and a small quantity was in dimers (Fig. 4). The average purity of Cip7-PTCBDengD after IMAC was 88.1 ± 5.8% (n = 5). 3.2.4. Purification of CBDcbpA -LG A higher purity of CBDcbpA -LG could not be achieved by using either AEC or IMAC alone so it was purified using a two-step process: AEC followed by IMAC. CBDcbpA -LG had a high starting purity of 37.2 ± 0.5% (n = 12). Most CBDcbpA -LG was eluted with NaCl at the conductivity of 15–30 mS. Peak for CBDcbpA -LG was obtained at 15–25 mS. The purity of CBDcbpA -LG after AEC increased from 37.2% to 43.8% (Fig. 5A). In IMAC, small amounts of CBDcbpA -LG were eluted at 50–75 mM imidazole and the peak was at 150–500 mM imidazole. A small protein (∼20 kDa) was co-purified with CBDcbpA LG and found to be not a CBDcbpA -LG degraded fragment by Western blotting analysis. An average purity of 86.2 ± 7.7% (n = 10) was obtained for CBDcbpA -LG after the two-step chromatography (Fig. 5B). 3.3. Binding of PTCBDengD and CBD-chimeras to Cuprophan powder

Fig. 3. Purification of LG-PTCBDengD from cell lysate using AEC. Lane 1: total protein from cell lysate; lane 2: flow through; lane 3: wash; lane 4: marker; lane 5: eluate at 7–9 mS; lane 6: eluate at 9–11 mS; lane 7: eluate at 11–11.5 mS; lane 8: eluate at 11.5–12.5 mS; lane 9: eluate at 12.5–13.5 mS; lane 10: eluate at 13.5–14.5 mS; lane 11: eluate at 14.5–15.5 mS; lane 12: eluate at 15.5–16.5 mS; lane 13: eluate at 16.5–17.5 mS; lane 14: eluate at 17.5–18 mS; lane 15: eluate at 18–19 mS; lane 16: eluate at 19–20 mS; lane 17: eluate at 20–21 mS.

The binding of PTCBDengD , Cip7-PTCBDengD , CBDcbpA -Cip7 and CBDcbpA -LG to amorphous regenerated cellulose Cuprophan was qualitatively observed and semi-quantitated by SDS-PAGE analysis. Cip7, LG-Doc, as well as E. coli proteins were used as negative controls for binding studies. The results showed that PTCBDengD , Cip7-PTCBDengD , CBDcbpA -LG and CBDcbpA -Cip7 specifically bound to Cuprophan powder (Fig. 6A–D). Negative controls Cip7, LG-Doc, as well as other E. coli proteins were observed not to bind (Fig. 6E). The purities of CBD-chimeric proteins before and after cellulose binding, and the purity after chromatography purification, was calculated and summarized in Table 4. Binding of CBDengD to crystalline cellulose Avicel has already been shown by Western blot analysis (Foong and Doi, 1992) and also in this study utilizing CBDengD fusions and crystalline cellulose Avicel (data not shown). However, the specific binding of CBDengD to regenerated, amorphous cellulose substrates has never been tested or analyzed. In this experiment, binding specificities

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Fig. 5. Purification of CBDcbpA -LG by two-step chromatography. (A) Purification of CBDcbpA -LG from cell lysate using AEC. Lane 1: total protein from cell lysate; lane 2: flow through; lane 3: wash; lane 4: eluate at 12.5–14 mS; lane 5: eluate at 14–15 mS; lane 6: eluate at 15–20 mS; lane 7: eluate at 20–25 mS; lane 8: eluate at 25–30 mS; lane 9: eluate at 30–32.5 mS; lane 10: marker. (B) Purification of CBDcbpA -LG from AEC eluate using IMAC. Lane 1: marker; lane 2: AEC eluate at 15–30 mS; lane 3: flow through; lane 4: wash; lane 5: eluate at 50 mM imidazole; lane 6: eluate at 75 mM imidazole; lane 7: eluate at 150 mM imidazole; lane 8: eluate at 500 imidazole.

Fig. 6. Specific binding of chimeras to Cuprophan. (A) Binding of PTCBDengD to Cuprophan. Lane 1: marker; lane 2: soluble cell lysate loaded on Cuprophan; lane 3: flow through; lane 4: 1st wash; lane 5: 2nd wash; lane 6: 3rd wash; lane 7: eluate. (B) Binding of Cip7-PTCBDengD to Cuprophan. Lane 1: marker; lane 2: soluble cell lysate loaded on Cuprophan; lane 3: flow through; lane 4: 1st wash; lane 5: 2nd wash; lane 6: 3rd wash; lane 7: 4th wash; lane 8: eluate. (C*) Appeared in Craig et al. (2007). Binding of CBDcbpA -LG to Cuprophan. Lane 1: marker; lane 2: soluble cell lysate loaded on Cuprophan; lane 3: flow through; lane 4: 3rd wash; lane 5: eluate. (D) Binding of CBDcbpA -Cip7 to Cuprophan. Lane 1: soluble cell lysate loaded on Cuprophan; lane 2: flow through; lane 3: 1st wash; lane 4: 2nd wash; lane 5: 3rd wash; lane 6: 4th wash; lane 7: eluate; lane 8: marker. (E) Binding of Cip7 and LG-Doc to Cuprophan. Lane 1: soluble cell lysate of Cip7 loaded on Cuprophan; lane 2: flow through; lane 3: 1st wash; lane 4: 3rd wash; lane 5: eluate; lane 6: marker; lane 7: soluble cell lysate of LG-Doc loaded on Cuprophan; lane 8: flow through; lane 9: 1st wash; lane 10: 3rd wash; lane 11: eluate; lane 12: marker.

Table 4 Purity of chimeric proteins before and after Cuprophan binding Chimera

PTCBDengD Cip7-PTCBDengD LG-PTCBDengD CBDcbpA -Cip7 CBDcbpA -LG

Purity (%) In soluble cell lysate

After Cuprophan binding

After chromatography purification

18.3 ± 0.8, n = 2 20.9 ± 4.5, n = 8 26.5 ± 3.0, n = 2 37.0 ± 3.7, n = 12 37.2 ± 0.5, n = 7

83.3 ± 3.2, n = 2 80.0 ± 7.4, n = 5 – 70.8 ± 2.9, n = 2 89.9 ± 0.8, n = 2

– 92.0 ± 0.7, n = 3 88.2 ± 3.1, n = 2 88.1 ± 5.8, n = 5 86.2 ± 7.7, n = 10

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of PTCBDengD and PTCBDengD -chimeric proteins to non-crystalline regenerated cellulose Cuprophan was demonstrated by the depletion of non-CBD proteins from E. coli in the bound fraction, which substantiates that it is a true CBD (Fig. 6A and B). The purity of PTCBDengD and Cip7-PTCBDengD after Cuprophan binding was 83.3% and 80.0%, respectively, which was lower but close to the purity of Cip7-PTCBDengD purified by IMAC 92.0% and also comparable to the purity of CBDcbpA -LG and CBDcbpA -Cip7 (Table 4). The results showed that the binding of PTCBDengD and PTCBDengD fusion protein was specific to Cuprophan and indicate that PTCBDengD can be used as a fusion/immobilization tag for purification on regenerated amorphous cellulose such as Cuprophan. The nucleotide sequence of EngD was determined and compared with the sequences of other cellulases (Hamamoto et al., 1992). It was found that the N-terminal catalytic region shared 80% homology with C. cellulovorans endoglucanase EngB and its C-terminal region including the PT linker had homology to Cellulomonas fimi exoglucanase Cex and endoglucanase CenA, as well as P. fluorescens endoglucanase and xylanase (Foong and Doi, 1992; Hamamoto et al., 1992). According to the sequence alignment results done by Hamamoto and colleagues as well as the specific binding results obtained in this study, it is deduced that the structure of EngD is similar to that of Cellulomonas fimi Cex (Poon et al., 2007). It is also hypothesized that CBDengD might have similar structure and binding mechanism to CBDCex . Specific binding of CBDengD -Cip7 and PTCBDengD -Cip7 to Avicel (results not shown) and Cip7-PTCBDengD to Cuprophan (Fig. 6B) showed that CBDengD retained its cellulose binding property when fused to Cip7 domain. Visual observations and analysis by SDS-PAGE analysis estimated that Cuprophan adsorbed more Cip7PTCBDengD than CBDcbpA -Cip7 (Fig. 6B and D). The difference in binding capacities may result from differences in their molecular sizes and the cellulose binding mechanisms of the CBDs. The stable binding of the 231-residue, 24 kDa CBDcbpA to cellulose requires at least 3 glucose chains alongside each other for anchoring and the planar linear strip of aromatic residues which plays a major role in interactive stacking to the cellulose chain requires six contiguous glucose rings in length (Tormo et al., 1996). On the other hand, the family 2a CBDengD can hypothetically bind to the cellulose chain glucose molecules utilizing three important aromatic residues (McLean et al., 2000). Therefore, more binding sites may be available for Cip7-PTCBDengD than CBDcbpA -Cip7. This may invariably make it better suited for binding to amorphous cellulose such as Cuprophan, which is an important material used in devices for dialysis, ultrafiltration and nutrient barrier applications. References Chatterjee, D.K., Esposito, D., 2006. Enhanced soluble protein expression using two new fusion tags. Protein Expr. Purif. 46, 122–129.

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Choi, S.K., Ljungdahl, L.G., 1996. Dissociation of the cellulosome of Clostridium thermocellum in the presence of ethylenediaminetetraacetic acid occurs with the formation of truncated polypeptides. Biochemistry 35, 4897–4905. Craig, S.J., Foong, F.C., Nordon, R., 2006. Engineered proteins containing the cohesin and dockerin domains from Clostridium thermocellum provides a reversible, high affinity interaction for biotechnology applications. J. Biotechnol. 121, 165– 173. Craig, S.J., Shu, A., Xu, Y., Foong, F.C., Nordon, R., 2007. Chimeric protein for selective cell attachment onto cellulosic substrates. Protein Eng. Des. Sel. 20, 235–241. Davis, G.D., Elisee, C., Newman, D.M., Harrison, R.G., 1999. New fusion protein systems designed to give soluble expression in E. coli. Biotechnol. Bioeng. 65, 382–388. Din, N., Gilkes, N.R., Tekant, B., Miller Jr., R.C., Warren, R.A.J., Kilburn, D.G., 1991. Nonhydrolytic disruption of cellulose fibres by the binding domain of a bacterial cellulose. Bio/technology 9, 1096–1099. Foong, F.C., Doi, R.H., 1992. Characterization and comparison of Clostridium cellulovorans endoglucanase-xylanases EngB and EngD hyperexpressed in Escherichia coli. J. Bacteriol. 174, 1403–1409. Goldstein, M.A., Takagi, M., Hashida, S., Shoseyov, O., Doi, R.H., Segel, I.H., 1993. Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. J. Bacteriol. 175, 5762–5768. Greenwood, J.M., Gilkes, N.R., Miller Jr., R.C., Kilburn, D.G., Warren, R.A.J., 1994. Purification and processing of cellulose-binding domain-alkaline phosphate fusion proteins. Biotechnol. Bioeng. 44, 1295–1305. Guan, C.di., Li, P., Riggs, P.D., Inouye, H., 1988. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltosebinding protein. Gene 15, 21–30. Hamamoto, T., Foong, F., Shoseyov, O., Doi, R.H., 1992. Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction. Mol. Gen. Genet. 231, 472– 479. Kapust, R.B., Waugh, D.S., 1999. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674. Kataeva, I., Guglielmi, G., Beguin, P., 1997. Interaction between Clostridium thermocellum endoglucanase CelD and polypeptides derived from the cellulosome-integrating protein CipA: stoichiometry and cellulolytic activity of the complexes. Biochem. J. 326, 617–624. LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.S., Schendel, P.F., McCoy, J.M., 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology 11, 187–193. Liu, Y., Zhao, T.J., Yan, Y.B., Zhou, H.M., 2006. Increase of soluble expression in Escherichia coli cytoplasm by protein disulfide isomerase gene fusion system. Protein Expr. Purif. 44, 155–161. McLean, B.W., Bray, M.R., Boraston, A.B., Gilkes, N.R., Haynes, C.A., Kilburn, D.G., 2000. Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues. Protein Eng. 13, 801–809. Murashima, K., Kosugi, A., Doi, R.H., 2003. Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulose EngD from Clostridium cellulovorans. Proteins 50, 620–628. Poon, D.K.Y., Withers, S.G., Mclntosh, L.P., 2007. Direct demonstration of the flexibility of the glycosylated proline-threonine linker in the Cellulomonas fimi xylanase Cex through NMR spectroscopic analysis. J. Biol. Chem. 282, 2091–2100. Smith, D.B., Johnson, K.S., 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31– 40. Tormo, J., Lamed, R., Chirino, A.J., Morag, E., Bayer, E.A., Shoham, Y., Steitz, T.A., 1996. Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. The EMBO J. 15, 5739–5751. Yeh, M., Craig, S., Lum, M.G., Foong, F.C., 2005. Effects of the PT region of EngD and HLD of CbpA on solubility, catalytic activity and purification characteristics of EngD-CBDCbpA fusions from Clostridium cellulovorans. J. Biotechnol. 116, 233– 244.