Use of Extracellular Medium Chain Length Polyhydroxyalkanoate

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Use of Extracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase for Targeted Binding of Proteins to Artificial Poly[(3-hydroxyoctanoate)-co-(3-hydroxyhexanoate)] Granules Julian Ihssen,† David Magnani,‡ Linda Tho¨ny-Meyer,† and Qun Ren*,† Laboratory of Biomaterials, Swiss Federal Laboratories for Materials Testing and Research (EMPA), CH-9014 St. Gallen, Switzerland, Roche Diagnostics AG, Advanced Systems Group, Forrenstrasse, CH-6343 Rotkreuz, Switzerland Received March 9, 2009; Revised Manuscript Received April 27, 2009

Polyhydroxyalkanoates (PHA), which are produced by many microorganisms, are promising polymers for biomedical applications due to their biodegradability and biocompatibility. In this study, we evaluated the suitability of medium chain length (mcl) PHA as surface materials for immobilizing proteins. Self-stabilized, artificial mclPHA beads with a size of 200-300 nm were fabricated. Five of six tested proteins adsorbed nonspecifically to mcl-PHA beads in amounts of 0.4-1.8 mg m-2 bead surface area. The binding capacity was comparable to similar-sized polystyrene particles commonly used for antibody immobilization in clinical diagnostics. A targeted immobilization of fusion proteins was achieved by using inactive extracellular PHA depolymerase (ePHAmcl) from Pseudomonas fluorescens as the capture ligand. The N-terminal part of ePhaZMCL preceding the catalytic domain was identified to comprise the substrate binding domain and was sufficient for mediating the binding of fusion proteins to mcl-PHA. We suggest mcl-PHA to be prime candidates for both nonspecific and targeted immobilization of proteins in applications such as drug delivery, protein microarrays, and protein purification.

Introduction Poly(3-hydroxyalkanoic acids) (PHA) are common carbon and energy storage polymers of bacteria. They receive increasing attention as natural resource-based, biodegradable alternatives to petrol-based polyolefins.1 PHA producing bacteria either synthesize short chain length (scl) PHA, for example, poly(3hydroxybutyrate) (PHB), or medium chain length (mcl) PHA, for example, poly(3-hydroxyoctanoate) (PHO), which differ considerably in their physicochemical properties.2 Bacterial PHA exhibit excellent biocompatibility and, thus, are promising polymers for biomedical applications such as drug delivery, implants, and tissue engineering.3 Various types of PHA are candidates for complementing poly(lactic acid), poly(glycolic acid), and poly(ε-caprolactone) materials currently used for these purposes.4 Adsorption of proteins to polymer surfaces is a key factor determining subsequent cell adhesion and thus the behavior of biomaterials in the human body.5,6 While it has been demonstrated that human blood proteins such as serum albumin and fibrinogen adsorb to poly(3-hydoxybutyrate-co-3-hydroxyvalerate) (PHBV),6 protein adsorption to mcl-PHA has not yet been investigated extensively. Certain proteins of PHB producing and degrading bacteria, for example, phasins and extracellular depolymerase, are known to bind to PHB and PHBV with high affinity.7-9 This property can be exploited for specific immobilization of fusion proteins onto scl-PHA.10-13 Similarly, putative binding domains of respective phasins have been used for selective binding of fusion proteins to native mcl-PHA granules inside of Pseudomonas putida cells.14 * To whom correspondence should be addressed. Phone: 41-71-2747688. Fax: 41-71-2747788. E-mail: [email protected]. † Swiss Federal Laboratories for Materials Testing and Research (EMPA). ‡ Roche Diagnostics AG.

Extracellular PHA depolymerases are either specific for sclPHA (ePhaZSCL) or for mcl-PHA (ePhaZMCL).15 Both types of depolymerases have been found in numerous bacterial species isolated from marine and terrestrial environments.16-19 Except for the typical “lipase box” motif G-X-S-X-G of serine hydrolases, as well as an aspartate and a histidine residue which form a catalytic triad together with the serine residue of the lipase box, ePhaZSCL and ePhaZMCL share no amino acid sequence homology.10,15,20 ePhaZSCL are organized in two distinct domains, an N-terminal catalytic domain and a Cterminal substrate binding domain, which are connected by a linker region.10,21-23 By contrast, in ePhaZMCL the lipase box and the catalytic triad residues are located in the C-terminal half of the protein,20 whereas the substrate binding domain is likely to be located in the N-terminal part of the prtotein, as deduced from the clustering of random mutations that affect PHA polymer degradation but not p-nitrophenyl-octanoate hydrolysis.24 In particular F83 and F96 seem to be important for mcl-PHA binding.25 Both ePhaZSCL and ePhaZMCL are secreted into the medium by PHA degrading bacteria and contain typical signal sequences of exoenzymes.20,26 Active ePhaZMCL can be used for microscale structuring of mcl-PHA surfaces, making use of differential hydrolysis rates of cross-linked and noncross-linked mcl-PHA.27 Further biocatalytic applications of ePhaZMCL such as enzymatic modification of polymer surface properties (similar to cutinases and lipases)28 or ester bond formation in organic synthesis have not yet been evaluated. This might be due to the lack of a high yield expression system for the production of recombinant ePhaZMCL. So far the enzyme has mostly been purified from wild-type PHA degrading bacteria cultivated in the presence of mcl-PHA.27,29 Although ePhaZMCL of Pseudomonas fluorescens GK13 was successfully cloned and recombinantly ex-

10.1021/bm9002859 CCC: $40.75  2009 American Chemical Society Published on Web 05/21/2009

Protein Immobilization on Artificial mcl-PHA Granules

Biomacromolecules, Vol. 10, No. 7, 2009

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Table 1. Plasmids Used in this Study plasmid pQE-30 pQE-70 pGEX-5x-3 pMALc2X pMALp2X pGFPuv pKS955 pMAD5 pMAD8 pMAD10 pMAD13 pMAD23 pJI41 pJI43 pJI44 pJI2 pJI14 pJI18 pJI31 pJI40

description

based on

vector for constructing cytoplasmic, N-terminal 6xHis fusions, Ptac, Ampr vector for constructing cytoplasmic, C-terminal 6xHis fusions, Ptac, Ampr vector for constructing cytoplasmic, N-terminal GST fusions, Ptac, lacI, Ampr vector for constructing cytoplasmic, N-terminal MBP2* fusions, Ptac, lacI, Ampr vector for constructing N-terminal MBP2* fusions secreted to the periplasm, includes MBP signal peptide, Ptac, lacI, Ampr gfp, Plac, Ampr PhaZS172A cytoplasmic GST-PhaZ35-278 (wild-type ePhaZMCL Pfl GK13 excluding native signal peptide) periplasmic MBP2*-PhaZ23-278 (wild-type ePhaZMCL PflGK13 excluding native signal peptide) periplasmic MBP2*-PhaZS172A23-278 (inactive variant of ePhaZMCL Pfl GK13) cytoplasmic MBP2*-PhaZ23-278 cytoplasmic MBP2*-PhaZS172A23-278 periplasmic MBP2*-PhaZ23-167 (N-terminal part of ePhaZMCL Pfl GK13 excluding signal peptide and active site) cytoplasmic MBP2*-PhaZ23-167 periplasmic MBP-PhaZ23-278 (wild-type ePhaZMCL, excluding native signal peptide, wild-type MBP fusion without MBP2* linker region) cytoplasmic 6H-GFP cytoplasmic GFP-6H PhaZ1-278-6H (wild-type ePhaZMCL Pfl GK13 including native signal peptide) cytoplasmic 6H-GFP-PhaZ23-278 cytoplasmic 6H-GFP-MBP-PhaZ23-278 (wild-type, mature MBP sequence inserted between GFP and PhaZ)

pressed in Escherichia coli, the volumetric yield remained low (≈5 mg L-1).20 In this study we evaluated whether ePhaZMCL of P. fluorescens GK13 can be used for targeted immobilization of fusion proteins onto artificial mcl-PHA granules. Both full-length, inactive ePhaZMCL S172A20 and the proposed N-terminal mcl-PHA binding part of ePhaZMCL were used for constructing fusion proteins. For comparison the nonspecific adsorption of proteins to mcl-PHA was also studied in detail. In addition, an improved system for recombinant expression of active ePhaZMCL was developed by making use of the solubility-enhancing properties of maltose binding protein.

Materials and Methods Bacterial Strains and Growth Conditions. Escherichia coli DH5R [supE44, ∆lacU169 (Ø80lacZ∆M15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1] was used for propagation of pJet/Blunt-derived cloning vectors and as expression strain for gluthathion-S-transferase (GST) fusion proteins. The malE negative E. coli strain ER2507 was used for expression of fusion proteins with pMAL-derived plasmids [genotype F-, ara-14, leuB6, fhuA2, ∆(argF-lac)U169, lacY1, glnV44, galK2, rpsL20(StrR), xyl-5, mtl-5 ∆(malB) zjc::Tn5(KanR), ∆(mcrC-mrr)] (New England Biolabs, Ipswich, U.S.A.). E. coli JM109 [gentotype endA1 recA1, gyrA96, thi, hsdR17 (rK-, mK+), relA1, supE44, λ-, ∆(lacproAB), (F′, traD36, proAB, lacIqZ∆M15)] (Promega, Madison, U.S.A.) was used as host for pQE-based plasmids in order to provide sufficiently high levels of LacI repressor. Except when noted otherwise, E. coli strains were grown at 37 °C on a shaker with 150 rpm in LB medium (5 g L-1 yeast extract, 10 g L-1 tryptone, 5 g L-1 sodium chloride) supplemented with 100 mg L-1 ampicillin. Pseudomonas fluorescens GK13 was grown in tryptic soy broth medium (Sigma, Buchs, Switzerland) at 30 °C. Construction of ePhaZMCL Fusion Proteins. Expression vectors used and plasmids constructed in this study are described in Table 1.

source Qiagen Qiagen Amersham/GE Healthcare New England Biolabs New England Biolabs

pBlue-script KSpGEX

BD Biosciences Clontech ref 20 this study

pMALp2x

this study

pMALp2x

this study

pMALc2x pMALc2x pMALp2x

this study this study this study

pMALc2x pMAD8 + pJI40

this study this study

pQE-30 pQE-70 pQE-70

this study this study this study

pJI2 pJI31

this study this study

Schematic views of ePhaZMCL fusion proteins used for PHO depolymerase and adsorption experiments are given in Figure 1. DNA sequences of primers used for amplifying genes and introducing restriction sites are available as supplementary online material. Genomic DNA of P. fluorescens GK13 (DSM 7139), plasmid pKS95520 and plasmid pGFPuv (BD Biosciences Clontech, Palo Alto, USA) served as templates for PCR amplification of wild-type ePhaZMCL, inactive variant ePhaZMCL PhaZS172A, and green fluorescent protein (GFP) DNA sequences, respectively. Genomic DNA of Escherichia coli DH5R was used as template for amplifying wild-type maltose binding protein (MBP). Genomic DNA of bacterial strains was purified using a commercially available kit (AquaPure genomic DNA isolation kit, BioRad, Hercules, U.S.A.). Plasmid DNA was purified with the columnbased GeneJet plasmid miniprep kit (Fermentas UAB, Vilnius, Lithuania). High-fidelity Phusion DNA polymerase (Finnzymes, Espoo, Finland) was used for PCR according to the manufacturers instructions, resulting in blunt-ended PCR products. PCR products were cloned into the vector pJet1/Blunt (Fermentas) by blunt end ligation. Inserts in plasmids were detected by restriction analysis and the cloned DNA sequences were verified (sequencing service: Synergene, Schlieren, Switzerland). Appropriate fragments were cut out from pJet1/Blunt constructs by double digestion and purified by gel extraction (MinElute columns, Quiagen, Valencia, U.S.A.). Plasmid pMAD5 was constructed by adding an EcoRI site at the 5′ end of the DNA sequence encoding wild-type ePhaZMCL starting from codon 35 (CGC ) arginine, N-terminal end of mature ePhaZMCL after secretion by P. fluorescens GK13) and an XhoI site at the 3′ end downstream of the native ePhaZMCL stop codon. The EcoRI-ePhaZMCL(aa 35-278-Stop)-XhoI fragment was fused in frame to the gluthathionS-transferase (GST) gene encoded by pGEX-5x-3, using the respective EcoRI and XhoI sites within the multiple cloning site (MCS) of the vector. Plasmids pMAD8 and pMAD13 were constructed by adding an EcoRI site at the 5′ end of the DNA sequence encoding wild-type ePhaZMCL starting from codon 23 (CGU ) alanine, N-terminal end of ePhaZMCL after secretion by recombinant E. coli) and a HindIII site at

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Figure 1. ePhaZMCL fusion proteins used for mcl-PHA degradation and adsorption experiments.

the 3′ end downstream of the native ePhaZMCL stop codon. The EcoRIePhaZMCL(aa 23-278-Stop)-HindIII fragment was fused in frame to the MBP gene and the linker region encoded by pMALc2x (cytoplasmic expression) and pMALp2x (periplasmic expression), using the respective EcoRI and HindIII sites within the MCS (see also Figure 1). Plasmids pMAD10 and pMAD23 were constructed accordingly, except that pKS955,20 encoding the inactive variant S172A of P. fluorescens GK13 ePhaZMCL was used as template for PCR. Plasmids pJI41 and pJI43 were constructed by adding an EcoRI site at the 5′ end of the DNA sequence encoding the N-terminal part of mature ePhaZMCL (A23Y167), which was arbitrarily chosen to end five codons before the codon for the first residue of the catalytic triad (S, Figure 1). A stop codon, a three nucleotide spacer and a HindIII site was added at the 3′ end of this DNA sequence. The EcoRI-ePhaZMCL(aa 23-167-Stop)-HindIII fragment was fused in frame to the MBP gene and the linker region encoded by pMALc2x and pMALp2x. In pMAL-derived plasmids, the C-terminal end of E. coli MBP lacks the last 4 amino acids (RITK) and is linked to the N terminal end of the fusion partner by a flexible linker region and a factor Xa protease cleavage site (Figure 1). If the EcoRI site of the pMAL MCS is used for cloning as in this study, the connecting sequence is NSSSNNNNNNNNNNLGIEGRISEF. In the following, the maltose binding protein with the linker extension encoded by pMALp/c2x-derived plasmids is abbreviated MBP2* as suggested by the manufacturer (New England Biolabs). Plasmid pJI2 was constructed by adding a SphI site at the 5′ end of the DNA sequence encoding full-length GFPuv and a KpnI site at the 3′ end, just before the native gfp stop codon (stop codons are already present in pQE vectors). The SphI-GFPuv(aa 1-239)-KpnI fragment was fused in frame with an N-terminal hexahistidine (6H) tag encoded by pQE-30, using respective SphI and KpnI sites within

the MCS. The construct resulted in the following extensions of the wild-type GFPuv amino acid sequence: MRGSHHHHHHGSAC at the N terminus and GTPGRPAAKLN at the C terminus (abbreviated 6HGFP). Plasmid pJI14 was constructed by adding a SphI site to the 5′ end of the DNA sequence encoding GFPuv. Thirty nucleotides of the MCS upstream of the GFVuv gene in plasmid pGFPuv were included in the construct. A BglII site was added to the 3′ end of the GFPuv gene, just before the native gfp stop codon. The SphI-MCS-GFPuv(aa 1-239)-BglII fragment was fused in frame with an N-terminal hexahistidine tag sequence of pQE-70, using the respective SphI and BglII sites within the MCS of pQE-70. The construct resulted in the following amino acid extensions of the wild-type GFPuv sequence: MLEDPRVPVE at the N terminus and RSHHHHHH at the C terminus (abbreviated GFP-6H). Plasmid pJI18 was constructed by creating a SphI site (GCATGC) around the start codon of the DNA sequence coding for the full-length, wild-type P. fluorescens GK13 ePhaZMCL gene and by adding a BglII site at the 3′ end, just before the native stop codon. The SphI-ePhaZMCL(aa 1-278)-BglII fragment was fused in frame with a C-terminal 6H tag sequence of pQE-70. The construct resulted in a C-terminal RSHHHHHH extension of the wild-type ePhaZMCL amino acid sequence (abbreviated PhaZ-6H). Plasmid pJI31 was constructed by adding a KpnI site at the 5′ end of the sequence coding for wild-type ePhaZMCL starting from codon 23 (A) and a HindIII site at the 3′ end. The KpnI-ePhaZMCL(aa 23-278)-HindIII fragment was fused in frame to the C-terminal end of 6H-GFP encoded by pJI2, using the respective SphI and BglII sites remaining from MCS of pQE30. In pJI31, GFPuv and ePhaZMCL23-278 are linked by a glycine and a threonine residue originating from the KpnI site, and the C terminal end of ePhaZMCL is extended by a KLN tripeptide. Plasmid pJI40 was constructed by adding KpnI sites at both the 5′ and 3′ ends

Protein Immobilization on Artificial mcl-PHA Granules of the DNA sequence coding for mature, wild-type E. coli MBP (K27 to K396). The KpnI-MBP(aa 27-396)-KpnI fragment was inserted in frame between the 6H-GFP and ePhaZMCL genes in pJI31, using the connecting KpnI site. In this construct, the C-terminal end of 6H-GFP is linked by a GT dipetide to the N terminal amino acid K27 of mature MBP, and the C-terminal end of MBP is again linked by GT to the N-terminal amino acid A23 of mature ePhaZMCL. Plasmid pJI44 was constructed by inserting a BglII-EagI fragment derived from pJI40 into pMAD8 double-digested with BglII and EagI. BglII cuts inside the malE (MBP) gene, while EagI cuts inside the phaZ gene. In the resulting construct (MBP-PhaZ23-278, Figure 1) MBP and ePhaZMCL are linked by only two amino acids (GT, from KpnI). Protein Purification and Analysis. For the production of recombinant proteins, shake flasks were inoculated with overnight LB cultures to an optical density (600 nm) of 0.05 and induced at an OD600 of 0.4-0.6 with 0.2 mM IPTG. Cells were harvested by centrifugation after an additional incubation of 4 to 10 h. Soluble intracellular proteins were released by a combined lysozyme-sonication treatment (native lysis): cell pellets were resuspended in amylose or HisTrap column loading buffer to an OD600 of 20, supplemented with 1 mg mL-l lysozyme, and were frozen at -20 °C. After rethawing, cell suspensions were incubated for 1 h at 37 °C. While cooled on ice, cells were subsequently disrupted by 10 ultrasonic pulses of 10 s, at 80% amplitude (Branson Sonifier, Danbury, U.S.A.). Cell debris was removed by centrifugation (13000 × g, 15 min). Periplasmic proteins were released by osmotic shock.30 Native lysis and osmotic shock supernatants were filtered through 0.2 µm PES membrane filters (Millipore, Bedford, ¨ KTA Prime U.S.A.) before applying them to purification columns. An A system equipped with an UV-vis detector was used for protein purification (GE Healthcare, Little Chalfont, U.K.). 6H-GFP was purified using a HisTrap FFcrude column (GE Healthcare) according to the standard protocol described in the manual for pQE vectors (Qiagen, Germantown, U.S.A.). MBP2* and MBP fusion proteins were purified according to the manufacturer’s instructions using an amylose column (New England Biolabs). For obtaining pure periplasmic MBP2*PhaZ23-278 and MBP2*-PhaZS172A23-278, eluates from the amylose column were dialyzed against phosphate-buffered saline and proteins were then separated by size exclusion chromatography (SEC; Superose 12, bed volume 100 mL, GE Healthcare) at a flow rate of 1 mL min-1. Protein concentrations in the range of 0.1 to 2 mg mL-1 were determined spectrophotometrically at 280 nm with a NanoDrop device (NanoDrop Technologies, Wilmington, U.S.A.). For each protein, extinction coefficients were calculated from the amino acid sequence using the ProtParam tool on the ExPASy server www.expasy.ch (e.g., GFP-6H E1% ) 7.51, MBP2* E1% ) 15.49). Protein concentrations in the range of 1-100 µg mL-1 were measured with the Bradford method using premixed reagent (Sigma, Buchs, Switzerland). Except for experiments with bovine serum albumin (BSA), bovine gamma globulin (BGG) was used as standard. For analysis of purity, molecular weight and relative band intensity of proteins in cell extracts, column eluates and defined protein mixtures, SDS-PAGE was performed using 10% acrylamide gels according to the method of Laemmli.31 Prior to electrophoresis, one volume of 2× SDS-PAGE sample buffer31 was added to one volume of protein solution and samples were heated for five minutes at 95 °C. For analysis of total cell proteins (TCP), cells were resuspended in 1× SDS-PAGE sample buffer to an OD600 of 10, and proteins were solubilized at 95 °C for 5 min. After electrophoresis, protein bands were visualized by Coomassie blue staining. Western blot analysis for the detection of maltose binding protein was performed according to standard procedures using RMBP antiserum from rabbit (New England Biolabs, Ipswich, U.S.A.) and a horseradish peroxidase (HRP)-coupled secondary goat-antirabbit antibody (Calbiochem-Merck, San Diego, U.S.A.). SuperSignal West Dura (Pierce Biotech, Rockford, U.S.A.) was used as HRP substrate for chemiluminescent detection.


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Preparation of mcl-PHA Suspensions (Artificial Granules). Poly[(3hydroxyoctanoate)-co-(3-hydroxyhexanoate)], for reasons of simplicity in the following abbreviated as PHO, was produced in dual carbonand nitrogen-limited chemostat cultures with Pseudomonas putida GPo1 as production strain and octanoic acid as sole carbon source. Growth medium, cultivation conditions, as well as extraction and purification procedures were as described previously.32 The purified polymer contained 86 mol % C8 monomers and 14 mol % C6 monomers, as determined by GC analysis.33 Self-stabilized suspensions of artificial PHO granules in double distilled water were prepared by the solvent evaporation method.20,34 Four volumes of PHO dissolved in acetone (2 g L-1) were quickly added to one volume of ice-cooled ddH2O while shaking. Subsequently, acetone was removed by rotary evaporation under vacuum at 35 °C. Final PHO concentrations in the milky suspensions were 7-9 g L-1. The quick addition of PHO solution to water resulted in sligthly larger granules (≈0.3 µm) than an average size of 0.09-0.2 µm as reported by Marchessault et al.34 PHO Depolymerase Activity and Protein Adsorption Assays. PHO depolymerase activity was measured at 30 °C as (i) para-nitrophenyloctanoate (p-NPO) esterase activity as described previously,19 and (ii) by decreasing turbidity of PHO suspensions at 650 nm.20 For quantification of p-nitrophenylate ions formed during p-NPO hydrolysis, an extinction coefficient ε of 14.93 mM-1 cm-1 at 405 nm was used.19 The absorbance ratio for the PHO suspension prepared in this study was determined to be 0.41 µL µg-1 cm-1. One unit of ePhaZMCL activity was defined as the amount of enzyme required for the hydrolysis of 1 µg PHO per minute.20 For qualitative evaluation of ePhaZMCL secretion, LB agar plates with a top layer containing PHO suspension were used.20 Protein binding to PHO was analyzed by incubating proteins in 100 mM Tris · HCl (pH 8.5) with 0.48 mgPHO mL-1 artificial PHO granules (similar PHO-water suspensions were used as suspensions used for the depolymerase assay). Proteins were added to final concentrations of 20 µg mL-1, corresponding to 42 µg protein per mg PHO. After specified incubation times at room temperature, PHO granules were removed by filtration (0.2 µm PES membranes) and the concentration nonadsorbed proteins was determined with the Bradford assay. In experiments with periplasmic MBP2*-PhaZS172A, PHO had to be removed by centrifugation at 20800 g for 10 min because granules with adsorbed periplasmic MBP2*- PhaZS172A passed through 0.2 µm PES membrane filters. Adsorbed amounts were calculated from the difference in protein concentrations between PHO-containing and PHO-free control tubes measured after incubation and filtration/ centrifugation. Competitive binding of ePhaZMCL fusions and other proteins was analyzed by incubating protein mixtures with artificial PHO granules for one hour at room temperature (final volume 200 µL, final concentration for each protein 50 µg mL-1, final PHO concentration 2.4 mg mL-1, 100 mM Tris · HCl, pH 8.5). After centrifugation for 10 min at 20800 g, 50 µL of the supernatant of PHO-containing and control tubes was removed for SDS-PAGE analysis. PHO pellets after centrifugation were washed twice by resuspension in 1 mL of 0.1 M Tris · HCl pH 8.5 followed by 30 min centrifugation at 20800 g. Proteins remaining adsorbed to the PHO pellet after washing were released by resuspending the pellet in 60 µL 0.1 M Tris · HCl pH 8.5 supplemented with 1% w/v sodium dodecylsulfate (Tris-SDS) and incubating the suspension for 45 min at room temperature () 3-fold concentration of released proteins with regard to the volume of initial solutions). After SDS treatment, PHO was removed by centrifugation (10 min at 20800 g) and 30 µL of the supernatant was withdrawn for SDS-PAGE analysis. Adsorption of proteins from E. coli native lysis supernatants and osmotic shock extracts was studied by adding 100 µL of PHO-water suspension to 200 µL of cell extract (final PHO concentration, 2.4 mg mL-1). Separation of PHO, washing and rerelease of adsorbed proteins was performed as described above for defined protein mixtures. Proteins adsorbed to PHO from cell extracts were rereleased in 200 µL TrisSDS (similar volume as the initial volume of the cell extracts). In the

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Figure 2. (A) Expression of various types of ePhaZMCL fusion proteins in recombinant Escherichia coli. TCP, total cell protein; NL, soluble proteins released by native lysis. Recombinant proteins recovered in soluble form are highlighted by boxes. (B) MBP2*-ePhaZMCL fusion proteins purified from osmotic shock fluid (lanes 1, 2, 3, and 6) and native lysate (lanes 4, 5, 7, and 8). (1) Periplasmic MBP2*-PhaZ23-278-amylose, (2) periplasmic MBP2*-PhaZ23-278-amylose and SEC, (3) periplasmic MBP-PhaZ23-278-amylose, (4) cytoplasmic MBP2*-PhaZ23-278-amylose, (5) cytoplasmic MBP2*-PhaZS172A23-278-amylose, (6 and 7) periplasmic MBP2*-PhaZ23-167-amylose, (8) cytoplasmic MBP2*-PhaZ23-167-amylose.

experiment with cell extract from plasmid-free E. coli ER2507, the adsorbed proteins were released in 60 µL SDS-Tris () 3-fold concentration) for better visualization. SDS-PAGE gels were loaded with 10 µL of denatured sample per well for experiments with defined protein mixtures and with 5 µL per well for experiments with cell extracts. Lyophilized beef heart cytochrome c and E. coli alkaline phosphatase (AP) were purchased from Sigma (Buchs, Switzerland), bovine gamma globulin (BGG) solution (2 mg mL-1) was obtained from Biorad (Reinach, Switzerland), bovine serum albumin (BSA) solution (2 mg mL-1) from Pierce Biotech, and MBP2* solution (6 mg mL-1) from New England Biolabs. Stock solutions of lyophilized proteins were prepared in ddH2O. Purchased protein solutions and proteins purified with amylose and HisTrap columns were buffer-exchanged to 100 mM Tris HCl (pH 8.5) or ddH2O by dialysis or ultrafiltration (YM-10 mini spin membrane columns, Millipore). Microscopy and Spectrophotometry. For visualizing bacterial cells and artificial PHO granules, a Leica DFC350 FX fluorescence microscope equipped with a black and white CCD camera was used (Leica Microsystems, Heerbrugg, Switzerland). Presence of correctly folded green fluorescent protein in recombinant E. coli cells was analyzed four hours after induction using an excitation wavelength of 450-490 nm and an emission filter of 525-550 nm. Exposure time for green fluorescence was 0.15-0.5 s. Artificial PHO granules in aqueous suspensions were stained by adding 1 µg mL-1 of the hydrophobic red fluorescent dye Nile red (Sigma). Nile red was excited at 515-560 nm and detected at g630 nm. Exposure time for red fluorescence was 20-40 ms. Average size of artificial PHO granules was determined graphically by measuring the particle diameter in pixels with the freeware graphic viewer IrfanView (www.irfanview.com) and multiplying this value with the calibrated pixel per µm ratio of the microscopic image. The changes of turbidity of PHO suspensions in depolymerase and binding assays were analyzed at a wavelength of 650 nm with a Genesys 6 spectrophotometer (Thermo electron, Cambridge, U.K.). Cuvettes containing the appropriate assay buffers were used for the automatically subtracted blind value.

Results Expression and Purification of ePhaZMCL Fusion Proteins. Previous studies demonstrated that the substrate binding domain of extracelluar scl-PHA depolymerase can be fused to the N-terminal end of either GFP, red fluorescent protein, or GST, resulting in fusion proteins which specifically bound to scl-PHA.10,11 We intended to construct analogous mcl-PHA

binding fusion proteins based on extracellular mcl-PHA depolymerase. For the construction of such fusions the use of either an inactive full-length ePhaZMCL variant, for example, S172A,20 or a not yet defined PHA binding domain can be envisaged, as both lack depolymerase activity. We speculated that the protein sequence of mature ePhaZMCL that precedes the S/D/H residues forming the catalytic triad, corresponding approximately to residues A23 to Y167, constitutes a putative mcl-PHA binding domain (Figure 1). To explore which type of fusion proteins can be expressed in a correctly folded and soluble form in E. coli, we constructed cytoplasmic expression fusions of ePhaZMCL to the N-terminal end of 6H-GFP, GST, and MBP2*, respectively. For comparison, hexahistidine-tagged ePhaZMCL and GFP were also constructed (Tab. 1). Upon induction with IPTG, specific bands of the expected sizes were detected in total cell extracts of E. coli harboring pJI2 (6H-GFP, theoretical molecular weight 29.0 kDa, Figure 2A, lane 2), pJI18 (PhaZ1-278-6H, expected size without cleaved signal peptide 28.9 kDa, Figure 2A, lane 4), pJI31 (6H-GFP-PhaZ23-278, 56.7 kDa, Figure 2A, lane 6), pMAD5 (GST-PhaZ35-278, 54.8 kDa, Figure 2A, lane 8), and pMAD13 (MBP2*-PhaZ23-278, 70.8 kDa, Figure 2A, lane 10). However, except for MBP2*-PhaZ23-278 and 6H-GFP (Figure 2A, lanes 3 and 11), none of the recombinant proteins could be recovered in soluble form by native cell lysis (Figure 2A, lanes 5, 7, and 9). In spite of the presence of the native P. fluorescens GK13 signal peptide for secretion to the periplasm, PhaZ1-278-6H formed insoluble intracellular aggregates in fully induced E. coli cells. Accordingly, no depolymerase activity could be detected in cell extracts. However, when the IPTG concentration was reduced to levels below 20 µM and the cultivation temperature was decreased to 30 °C, secretion of active PhaZ1-278-6H could be detected on PHO indicator plates as transparent halos around colonies (results not shown). As expected, E. coli cells expressing soluble 6H-GFP and GFP-6H (pJI14) exhibited strong green fluorescence. By contrast, and in agreement with the formation of misfolded, insoluble cytoplasmic aggregates, cells expressing 6H-GFPPhaZ showed no green fluorescence. N- and C-terminal GFP fusions with the suspected mcl-PHA binding part of ePhaZMCL (PhaZ23-167-GFP-6H and 6H-GFP-PhaZ23-167) were also expressed to high levels in the cytoplasm of E. coli but remained completely insoluble (data not shown). Again, no green fluorescence could be detected in induced cells.



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Table 2. Specific Depolymerase Activities of Cell Extracts and (Partially) Purified MBP2*-PhaZMCL Fusion Proteinsa fusion protein periplasmic MBP2*-PhaZ23-278 periplasmic MBP2*-PhaZ23-278 periplasmic MBP2*-PhaZ23-278 periplasmic MBP-PhaZ23-278 periplasmic MBP2*-PhaZS172A23-278 cytoplasmic MBP2*-PhaZ23-278 periplasmic MBP2*-PhaZ23-278 a

purification

p-NPO esterase (U mg-1)

osmotic shock 0.03 ((0.007) amylose 0.23 ((0.05) amylose - SEC 0.78 ((0.20) amylose 0.18 ((0.03) amylose 0 amylose 0 0.05% v/v Tween 20 amylose 0

PHO depolymerase suspension method (µgPHO min-1 mgprotein-1) 7.9 ((0.4) × 103 13.5 ((3.4) × 103 39.9 ((7.0) × 103 3.9 ((0.7) × 103 0 0 0

Average values and standard deviations for n ) 3 replicate assays.

Fusion proteins with ePhaZMCL were constructed beginning with A23 because this residue was identified as the N-terminal end of recombinant ePhaZMCL after secretion by E. coli.20 A23 is preceded by an A-X-A motif typical for a signal peptide cleavage site of proteins exported to the periplasm via the Sec pathway in E. coli.35 We also attempted to construct fusion proteins starting with R35, one of two reported N-termini of ePhaZMCL when secreted to the medium by P. fluorescens GK13.20 However, except for GST-PhaZ35-278 (Figure 2A, lane 8) none of the constructs yielded a detectable band for the recombinant proteins when expressed in E. coli (data not shown). Similarly to MBP2*-PhaZ23-278 (Figure 2A, lane 11), MBP2*PhaZS172A23-278 (pMAD13) and MBP2*-PhaZ23-167 (pJI43) were expressed in soluble form in the cytoplasm of E. coli. In addition to cytoplasmic fusion proteins, periplasmic MBP2* fusions with ePhaZMCL were constructed to evaluate the effect of the cellular compartment of expression on mcl-PHA depolymerase activity and mcl-PHA binding. For both MBP2*PhaZ23-278 (pMAD8) and MBP2*-PhaZS172A23-278 (pMAD23) strong bands corresponding in size to secreted fusion protein ()70.8 kDa) could be detected in osmotic shock fluid (see also Figure 5A, lane OS). Soluble ePhaZMCL fusion proteins were purified with an amylose column, making use of the specific binding ability of MBP. While cytoplasmic MBP2*-PhaZ23-278, MBP2*PhaZS172A23-278, and MBP2*-PhaZ23-167 eluted as a single band (Figure 2B, lanes 4, 5, and 8), periplasmic MBP2*PhaZ23-278 and MBP2*-PhaZS172A23-278 contained equal amounts of a contaminating protein (Figure 2B, lane 1). This protein was identified as MBP2* by Western blot analysis (data not shown). Periplasmic MBP2*-PhaZ and MBP2*-PhaZS172A could be separated from MBP2* by size exclusion chromatography (Figure 2B, lane 2). However, due to incomplete separation only a small fraction of the total amount of the fusion protein could be recovered in pure form. The flexible linker region and the factor Xa protease site of MBP2* might contribute to cleavage of MBP2*-PhaZ by E. coli proteases in the periplasm. However, an MBP-PhaZ23-278 fusion lacking this region (Figure 1) also coeluted with cleaved bands in the size range of MBP (Figure 2B, lane 3). Periplasmic MBP2*PhaZ23-167 (pJI41) yielded exclusively MBP2* when purified from osmotic shock fluid (Figure 2B, lane 6). A faint band corresponding in size to MBP2*-PhaZ23-167 with signal peptide (61.2 kDa) was detected when whole cell lysate was used for purification, indicating that cleavage occurs during or after translocation to the periplasm (Figure 2B, lane 7). PHO Depolymerase Activity of ePhaZMCL Fusion Proteins. Periplasmic MBP2*-PhaZ23-278 exhibited both p-NPO esterase and depolymerase activity in the PHO suspension turbidity assay (Tab. 2). As expected, specific activities increased in the following order: osmotic shock fluid < amylose eluate < SEC-

purified MBP2*-PhaZ23-278 (Table 2). Calculated from activities in osmotic shock extracts, the volumetric yield of mcl-PHA depolymerase in LB shake flask cultures was 1742 ((77) × 103 U L-1, corresponding to 44 mg L-1 MBP2*-PhaZ23-278. Depolymerase activity in amylose eluates containing periplasmic MBP2*-PhaZ23-278 remained stable for several months at 4 °C. Omitting the 24 amino acid flexible linker region of MBP2* did not abolish depolymerase activity in the secreted MBPPhaZ23-278 fusion; however, the specific activity was reduced approximately 3-fold compared to MBP2*-PhaZ23-278 (Table 2). When MBP2*-PhaZ23-278 was expressed in the cytoplasm of E. coli, the purified fusion protein was completely inactive (Table 2). To evaluate whether the lack of activity of cytoplasmic MBP2*-PhaZ23-278 was due to missing disulfide bonds, periplasmic MBP2*-PhaZ23-278 was incubated with 100 mM dithiothreitol for 30 min to reduce any disulfide bonds formed after secretion. However, no negative effect on depolymerase activity was observed (data not shown). By contrast, PHO depolymerase and p-NPO esterase activity of periplasmic MBP2*- PhaZ23-278 was totally lost in the presence of 0.05% v/v of a mild detergent (Table 2). Protein-Induced Aggregation of PHO Granules. In negative controls for the PHO depolymerase assay it was found that addition of MBP2* or other proteins to aqueous PHO suspension led to an increase in turbidity. This phenomenon was investigated in more detail. Upon protein addition, OD650 increased in a time-dependent manner, and depending on the type of protein used, a stable, higher OD650 value was reached within five to 60 min (data not shown). The change in final OD650 in 0.1 M Tris · HCl buffer at pH 8.5 varied between zero (alkaline phosphatase, AP) to a 7-fold increase (cytochrome c; Figure 3A). In the case of MBP2*, the presence of low concentrations of sodium chloride (20 mM) resulted in a higher final OD650 (Figure 3A). At concentrations above 250 mM, sodium chloride by itself caused an increase in OD650 (final OD650 ) 0.9-1.0). The changed optical properties of the PHO suspensions were due to a substantially increased average granule size (Figure 3B). At OD650 ≈ 0.2 the average granule diameter was 0.28 ( 0.07 µm (n ) 95), while it was increased to 0.88 ( 0.29 µm (n ) 44) at OD650 ≈ 1.2 in a suspension containing a similar amount of PHO per volume. In high OD650 suspensions, granule number per volume was decreased markedly (Figure 3B), which indicates that the larger granules emerged by aggregation or coalescence of small granules. While addition of MBP2* induced the formation of aggregates of several small granules (Figure 3B), cytochrome c (Figure 3B), BSA and GFP-6H caused the formation of larger-sized circular granules. The detergent Tween 20 efficiently prevented protein-induced aggregation of PHO granules (Figure 3A).

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Figure 3. (A) Effect of the addition of proteins on the optical density of PHO suspensions (0.48 mgPHO mL-1). Proteins were added in concentrations of 20 µg mL-1 each and OD650 was determined after incubation for 1 h at room temperature: empty bar, no NaCl was added; filled bar, 20 mM NaCl was added. (B) PHO granule size at different optical densities (red fluorescent images after staining with nile red).

Although an interaction of ePhaZMCL domains with the surface of PHO granules can be expected, the presence of (inactive) periplasmic MBP2*-PhaZS172A23-278 prevented aggregation of PHO granules (Figure 3A,B). MBP2*-PhaZS172A stabilized PHO suspensions even if several times more aggregationinducing proteins were added (Figure 3A,B). Adsorption of Proteins to Artificial PHO Granules. Due to the observed nonspecific interaction of various proteins with PHO suspensions, the adsorption of six proteins, ranging in size from 12.3 kDa (cytochrome c) to 66.4 kDa (BSA), to artificial PHO granules was quantified. Adsorbed amounts given in Table 3 were quantified from the decrease in protein concentration after PHO addition divided by the specific surface area of added PHO granules. Assuming a density (F) of 1.05 g cm-3 for PHO36 and using an average diameter (d) of 0.28 µm determined from microscopic images, the specific surface area (am) of nonaggregated, spherical PHO granules was calculated to be 20.3 × 10-3 m2 mgPHO-1, using the formula am ) 6/F/d. The amount of protein adsorbing to PHO granules varied 4.5-fold for different proteins and ranged from 0.4 mg m-2 (cytochrome c) to 1.8 mg m-2 (BGG) (Table 3). No significant adsorption was

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detected for alkaline phosphatase (Table 3). Except for GFP6H which dissociated again from the granules after an initial maximal adsorption of 2.3 mg m-2, no pronounced changes in the amount of adsorbed protein were observed within a three hour incubation period (Table 3). The observed slight decrease could be caused by equilibrium of absorption and deassociation of proteins. As expected periplasmic and cytoplasmic MBP2*PhaZS172A23-278 also adsorbed to artificial PHO granules. The amount of adsorbed protein per surface area was in the same order of magnitude as for MBP2* and other nonspecifically adsorbing proteins (Table 3). Degradation of PHO in the Presence of Nonspecifically Adsorbing Proteins. To test whether active ePhaZMCL has a higher affinity for PHO than nonspecifically adsorbing proteins, active, periplasmic MBP2*-PhaZ23-273 was incubated with PHO suspensions together with other proteins. When 20 µg mL-1 amylose-purified periplasmic MBP2*-PhaZ23-273 (containing equal amounts of MBP2*) was added to PHO suspension, OD650 decreased to zero within 7 min (Figure 4). When the PHO suspension was preincubated with alkaline phosphatase, which does not adsorb to PHO, no difference in degradation kinetics was observed upon addition of periplasmic MBP2*-PhaZ23-273 (Figure 4). The presence of four additional proteins (20 µg mL-1 each), which have the capability to adsorb to PHO, slowed down, but did not prevent hydrolysis of PHO granules (Figure 4). PHO suspensions preincubated for 30 min with either MBP2* or BGG could also be degraded by MBP2*-PhaZ23-273 (Figure 4), in spite of the fact that the surface must have already been covered by a protein layer. Again, degradation rate was reduced. Preferential Adsorption of ePhaZMCL Fusion Proteins to PHO. The capability of full-length or partial ePhaZMCL fusion proteins to preferentially adsorb to artificial PHO granules was analyzed in competitive binding assays in order to evaluate whether inactive ePhaZMCL can be used for targeted immobilization of fusion proteins. When PHO suspension was added to either native lysate (total soluble proteins) or osmotic shock fluid of recombinant E. coli expressing periplasmic MBP2*PhaZS172A23-167, specific binding of the fusion protein to PHO was observed as demonstrated by the pattern of proteins released by SDS from washed PHO granules (Figure 5A). Similarly, cytoplasmic () inactive) MBP2*-PhaZ23-278 and MBP2*PhaZS172A23-278 preferentially adsorbed to PHO from E. coli cell extracts (Figure 5A). It was also found that the N-terminal part of ePhaZMCL, comprising amino acids A23 to Y167, was sufficient for conferring PHO binding specifity to a cytoplasmic MBP2* fusion protein (Figure 5A). Numerous proteins of E. coli adsorbed to PHO when native lysate of plasmid-free E. coli ER2507 was incubated with a PHO suspension (Figure 5A). The relative band intensities of E. coli proteins released from PHO differed clearly from the relative band intensities in the original cell extract (Figure 5A). In competitive binding assays with defined mixtures of proteins, weakening of bands after incubation with artificial PHO granules and concomitant release of adsorbed proteins from washed granules was analyzed. The band for periplasmic MBP2*-PhaZS172A23-278 in amylose eluate containing equal amounts of MBP2* was weakened after incubation with PHO, while band intensity of MBP2* remained similar (Figure 5B). In agreement with this, after release of adsorbed proteins by SDS, only a band for MBP2*-PhaZS172A23-278 appeared, in spite of the fact that MBP2* readily adsorbed to and could be released from PHO when present alone (Figure 3, Figure 5B).



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Table 3. Amount of Protein Adsorbing per Surface Area of Artificial PHO Granules (Added Concentration of PHO ) 0.48 mg mL-1)a amount adsorbed to PHO granules (mg m-2) protein MBP2* BSA GFP-6H BGG cytochrome c alkaline phosphatase periplasmic MBP2*-PhaZS172A23-278 cytoplasmic MBP2*-PhaZS172A23-278 a

5 min 0.87 ((0.07) 0.85 ((0.13) 2.25 ((0.04) 1.87 ((0.01) 0.48 ((0.09) 0 0.86 ((0.03) 0.66 ((0.05)

60 min 0.76 ((0.1) 0.67 ((0.15) 1.30 ((0.05) 1.86 ((0.06) 0.62 ((0.17) 0 n.a. 0.60 ((0.05)

3h 0.67 ((0.03) 0.63 ((0.08) 0.46 ((0.08) 1.77 ((0.08) 0.38 ((0.12) 0 n.a. 0.59 ((0.06)

Average values and standard deviations for n ) 3 replicate experiments; n.a.: not analysed.

Figure 4. Degradation of PHO (suspension with 0.48 mgPHO mL-1) at pH 8.5 with and without preadsorbed proteins by active MBP2*ePhaZMCL. Proteins were added in concentrations of 20 µg mL-1 each. Assays were performed at room temperature.

Similarly, after incubation with artificial PHO granules the band for cytoplasmic MBP2*-PhaZS172A23-167 selectively disappeared from a mixture containing three nonspecifically adsorbing proteins (Figure 5B). A certain amount of BSA coadsorbed from the mixture, but the strongest band after release of proteins from washed PHO granules was clearly MBP2*-PhaZ23-167 (Figure 5B). It is possible that the surface of the granules was not completely saturated by MBP2*-PhaZ23-167, facilitating the adsorption of a certain amount of BSA. In agreement with the data presented in Table 3, SDS-PAGE analysis showed that both MBP2* and BGG were captured by added PHO and could be rereleased from washed granules by SDS (Figure 5B). In contrast to washing with Tris buffer, washing with buffer containing 0.05% v/v Tween 20 (a detergent considered as “mild”) removed most of the adsorbed MBP2*-PhaZS172A23-278 as well as MBP2* (Figure 5B).

Discussion High Yield Expression System for mcl-PHA Depolymerase. In this study an efficient expression system for the production of active mcl-PHA depolymerase in E. coli was established by fusing ePhaZMCL of P. fluorescens GK13 to the C-terminal end of MBP2*. Taking into account a 62% contribution of MBP2* to the molecular weight of the fusion protein, specific p-NPO

esterase activity of ePhaZMCL expressed as MBP2*-PhaZ23-278 was 2.1 U mg-1, which is close to 1.6-1.7 U mg-1 reported for purified ePhaZMCL from wild-type mcl-PHA degrading bacteria.19,29 We were able to increase the volumetric yield of recombinant mcl-PHA depolymerase in LB shake flasks 6-fold [from 277 × 103 U L-1 (achievable with pKS74420) to 1741 × 103 U L-1 (obtainable with pMAD8)]. Purity and volumetric activity of MBP2*-ePhaZMCL solutions obtained by simple osmotic shock treatment should already be sufficient for biocatalytic applications. Potential applications of recombinant MBP2*-ePhaZMCL are, among others, surface modification of non-PHA polyesters,28,37 microscale structuring of partially cross-linked mcl-PHA,27 enzymatic resolution of chiral compounds,38 or ester synthesis in nonaqueous media.38 Using MBP2*-ePhaZMCL instead of wild-type ePhaZMCL in enzymatic processes might have additional benefits such as decreasing protein precipitation or irreversible adsorption of the enzyme to hydrophobic surfaces. The improved recombinant expression system also allows further engineering of ePhaZMCL by site directed mutagenesis or directed evolution. The observed tendency of ePhaZMCL to form insoluble inclusion bodies when overexpressed alone or as fusion protein with GFP or GST is presumably due to the hydrophobic nature of the protein. Mature ePhaZMCL contains 33.2% amino acids with side chains defined as hydrophobic (Leu, Ile, Phe, Trp, Tyr, Val)39 and is characterized by a high proline content of 8.6% which also may impair soluble expression in E. coli. By contrast, highly soluble 6H-GFP contains 28.8% hydrophobic amino acids and only 4.7% proline. N-terminally fused MBP was found to strongly increase solubility of ePhaZMCL in recombinant E. coli. The solubilityenhancing properties of maltodextrin-binding proteins have become more and more evident in recent years.40 Solubility enhancers are thought to play a passive role in stabilizing and assisting proper folding of hydrophobic passenger proteins rather than directly acting as a chaperone.41 In contrast to ePhaZSCL,42 no crystal structure is yet available for ePhaZMCL. ePhaZMCL shares no sequence homology to PhaZSCL except for the catalytic triad residues.24 MBP fusions with short linkers of 3-5 amino acids have successfully been used for crystallizing hydrophobic proteins.43 A similar strategy could now be employed for ePhaZMCL because a periplasmic MBP-PhaZ23-273 fusion linked by only two amino acids (pJI40) was constructed in this study and proved to possess PHO depolymerase activity. Secretion of ePhaZMCL to the Periplasm is Required for Activity but not for mcl-PHA Binding. Many exoenzymes require formation of disulfide bonds for attaining full functionality; in E. coli this is achieved by the oxidizing periplasmic protein DsbA.44 In this study, secretion to the periplasm was found to be required for obtaining active MBP2*-ePhaZMCL. Although the primary amino acid sequence of ePhaZMCL of P. fluorescens GK1320 contains four cysteine residues which

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Figure 5. (A) Adsorption of proteins to artificial PHO granules from cell extracts of fusion protein-expressing wild-type E. coli. NL, native cell lysate; OS, osmotic shock fluid; PHO, proteins released from washed PHO pellets with 1% SDS. (B) Adsorption of proteins to artificial PHO granules from defined protein mixtures. Ctr, control without added PHO; PHO, samples incubated with artificial PHO granules; SN, supernatant after removal of PHO granules; P, proteins released from washed PHO pellets with 1% SDS; P*, proteins released from PHO pellets by SDS after two times washing with 0.05% Tween 20.

potentially could form disulfide bonds, the requirement for periplasmic expression to obtain active enzyme is unlikely to be due to disulfide bonds because ePhaZMCL enzymes are generally not inhibited by reducing agents,38,45 which was also the case for MBP2*-ePhaZMCL in this study. In spite of the lack of PHO depolymerase activity, at least the MBP part of intracellularly expressed MBP2*-ePhaZMCL must have folded correctly because the fusion protein obviously bound to amylose resin. It is possible that certain chaperones present only in the periplasm of E. coli, for example, SurA, FkpA, or OmpH,46 are required for correct formation of the tertiary structure of ePhaZMCL. Interestingly, (inactive) cytoplasmic MBP2*-

Ihssen et al.

PhaZ23-278 preferentially adsorbed to PHO granules from protein mixtures, which was also observed for cytoplasmic MBP2*PhaZS172A23-278 and MBP2*-ePhaZ23-167 (Figure 5A,B). Thus, it seems that the domains responsible for polymer binding attained their correct conformation also in the cytoplasm, while the catalytic triad responsible for ester bond cleavage did not form properly. In contrast to ePhaZMCL, it is obvious both from the structure and from the inhibitory effect of reducing agents that ePhaZSCL require disulfide bond formation in the periplasm for activity.24,42,47 Yet, cytoplasmic GST and GFP fusions of the substrate binding domain of ePhaZSCL adsorbed to scl-PHA with high affinity as well.10,11,23,48 Unfortunately, in neither of these studies a full-length cytoplasmic ePhaZSCL fusion was constructed and tested for activity. Theoretically such a fusion should be inactive, similarly to cytoplasmic MBP2*-ePhaZMCL. Interrelation of Protein Adsorption and the Stability of PHO Suspensions. It was found in this study that proteins destabilize suspensions of artificial PHO granules. Unamended suspensions of artificial PHO granules in H2O remain stable for up to two years,34 presumably because repulsive, electrostatic forces of surface carboxyl groups neutralize attracting van der Waals forces between particles.49 In general, colloidal stability decreases with increasing electrolyte concentration,49 which was also observed for PHO suspensions in this study. In the case of chloromethyl styrene latex particles with a comparable size of ≈0.2 µm, critical coagulation concentrations for sodium chloride of around 300 mM were reported,49 which is in agreement with NaCl concentrations g250 mM, causing aggregation of PHO granules. The adsorption of proteins induced aggregation of PHO granules already at low salt concentrations (Figure 3). In agreement with these findings, adsorption of immunoglobuline G caused a reduction in the critical coagulation concentration for chlormethyl styrene latex to 30 mM Na+.49 Particle aggregation due to the adsorption of proteins can be caused by several mechanisms, for example, bridging flocculation mediated by positively charged protein molecules50 or conformational changes of proteins during adsorption, leading to surface exposure of hydrophobic side chains, which in turn favor further hydrophobic interactions.51,52 Interestingly, adsorption of periplasmic MBP2*-PhaZS172A prevented protein-induced aggregation of PHO granules. Presumably, the MBP2* part of MBP2*-PhaZS172A is outcompeted from adsorption by preferential binding of the ePhaZMCL domains. MPB2* being kept in a native, outward oriented state might in turn preserve repulsive electrostatic forces between particles in spite of the adsorbed protein layer. Thus, MBP2*PhaZS172A constitutes an amphiphilic macromolecule with a hydrophobic (ePhaZMCL) and a hydrophilic (MBP2*) part, much like phasin proteins that also prevent native, intracellular PHA granules from coalescing.7,14,53 The nonionic, amphiphilic surfactant Tween 20 also effectively prevented protein-induced PHO granule aggregation, most likely because adsorbed detergent molecules shielded the PHO surface from interactions with proteins. Tween 20 even hindered active MBP2*-PhaZ23-278 from binding and hydrolyzing PHO granules (Figure 3). This in agreement with a 90% reduction in PHO depolymerase activity in the presence of 0.1% Tween 20 reported for ePhaZMCL from Pseudomonas alcaligenes M4-754 and with the inhibitory effect of other nonionic detergents on various ePhaZMCL.45 PHO Latex is Well-Suited for Nonspecific Protein Immobilization. Suspensions of self-stabilized PHO granules can be considered as a type of latex,34 similarly to dispersed


polystyrene particles.55 Except for alkaline phosphatase, all tested proteins unspecifically adsorbed to PHO latex in this study. Few published data on the adsorption of proteins to mclPHA were found in the literature.54 However, for chloromethylstyrene latex particles with a comparable average diameter of 0.2 µm, protein adsorption per surface area was in a similar range from 0.05 mg m-2 (BSA) to 4 mg m-2 (fibrinogen) at pH 849 and from 0.6 mg m-2 (lysozyme, ribonuclease) to 1.4 mg m-2 (R-lactalbumin) at pH 7.56 Similarly to the comparably high amounts of BGG (a mixture of immunoglobulins) found to adsorb to PHO latex, 6-fold more human immunoglobulin G adsorbed to chloromethylstyrene latex than, for example, myoglobin.49 The observed completion of protein adsorption onto PHO latex within 5 min also is in agreement with published data for polystyrene-coated silica particles and poly(methyl methacrylate) surfaces, where the amount of adsorbed protein leveled off after 80 and 200 s, respectively.56,57 The reluctance of alkaline phosphatase (AP) to adsorb to PHO cannot be explained by charge differences at pH 8.5 because the pI of AP (5.3) is in between values for adsorbing proteins such as MBP2* (4.8) and GFP-6H (6.6). However, the relative proportion of amino acids with hydrophobic side chains is markedly lower for AP (22.2%) compared to MBP2* (29.2%), BSA (28.2%), and GFP-6H (28.8%). If adsorption to PHO latex is dominated by hydrophobic interactions, this could be an explanation for the difference. Alternatively, a relatively high stability of the native AP conformation could be the cause for the observed differences as structural stability of proteins is another important factor determining surface adsorption.56 The striking differences in the pattern of PHO-adsorbed versus total soluble proteins of E. coli (Figure 5B) also suggest that differences in hydrophobicity, structural stability and other physicochemical properties influence adsorption to PHO latex. Antibodies immobilized on polystyrene particles are commonly used for diagnostic immunoassays in so-called latex agglutination tests.55 Similar applications can be envisaged for mcl-PHA latex given the high capacity for BGG adsorption. Furthermore, artificial PHO granules sensitized with antibodies could be used for drug delivery to specific target cells in the human body. IgG-coated poly(D,L-lactic acid/glycolic acid) particles in the size range of artificial PHO granules have been suggested as ideal drug nanocarriers with targeting abilities.58 An advantage of mcl-PHA carriers would be a high degree of biocompatibility combined with degradation via (slow) surface erosion.3 In contrast to this, poly(lactic acid) may undergo swelling and fast (burst) hydrolysis. Furthermore, mcl-PHA offer the possibility to tailor polymer properties by feeding different monomeric substrates to the production strain,59 which facilitates the engineering of surface characteristics and protein adsorption behavior of mcl-PHA granules to specific needs. Recently it was demonstrated that artifcial PHBV granules prepared with the help of polyvinylalcohol can be coated with scl-PHA-binding PhaP fusion proteins, which in turn directed the particles to specific disease tissues in a mouse model.13 An mcl-PHA based system would have two advantages compared to the described PHBV system: (i) no detergents are needed for the preparation of artifical granules,34,60 (ii) proteins (e.g., antibodies) can be immobilized without the help of a bacterial amino acid sequence which may induce an unwanted immune response. ePhaZMCL has a High Affinity for PHO and can be Used for Targeted Immobilization of Fusion Proteins. In this study, several lines of evidence for ePhaZMCL having a higher affinity for adsorption to mcl-PHA surfaces than other proteins are presented. First, it was shown that active MBP2*-ePhaZMCL was


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able to displace preadsorbed protein molecules from the PHO surface and initiate PHO hydrolysis. Second, inactive MBP2*ePhaZS172A prevented aggregation of artificial PHO granules in the presence of proteins that adsorb to PHO and induce granule aggregation when present alone. Third, periplasmic and cytoplasmic MBP2*-PhaZS172A preferentially and selectively adsorbed to artificial PHO granules from complex as well as defined mixtures of proteins in competitive binding assays. The observation that MBP2*-PhaZ23-167, which contains only the N-terminal part of mature ePhaZMCL was selectively captured by PHO from protein mixtures shows that this part of the protein comprises the mcl-PHA binding domain, as has been suggested previously from mutant analysis.38 Furthermore, preferential binding of MBP2*-PhaZ23-167 to artificial PHO granules demonstrates that the N-terminal substrate binding domain(s) of ePhaZMCL can be used as affinity tag for immobilization and purification of recombinant proteins based on processed mclPHA. A similar approach for immobilizing proteins on processed scl-PHA by the help of the substrate binding domain of ePhaZSCL has been reported previously.61,11 Protein immobilization mediated by biospecific tags is superior to random protein adsorption because it provides for oriented immobilization, preventing the solution-oriented passenger proteins from unfolding which often occurs during nonspecific adsorption.62,63 The N-terminal part of PhaF (a phasin) was used as a tag for capturing fusion proteins on native mcl-PHA granules inside of P. putida cells.14 However, in contrast to the ePhaZMCL tag this system is restricted to native granules inside of mcl-PHA producing microbes. Furthermore, with the PhaF system, coimmobilization of other PHA-associated proteins such as PhaC, intracellular PhaZ and PhaI cannot be prevented. A feature of the ePhaZMCL tag that still needs to be improved is the tendency of ePhaZMCL fusion proteins to form insoluble aggregates inside E. coli. This problem might be solved by including suitable linkers in the construct, by reducing the length of the ePhaZMCL tag to the minimum required for mcl-PHA binding or by directed evolution of the ePhaZMCL amino acid sequence with the aim of finding more soluble variants. Another possibility would be to use other expression hosts, for example, Ralstonia eutropha or Bacillus subtilis. Acknowledgment. We wish to thank Dr. Manfred Zinn, Ernst Pletscher, and Thomas Ramsauer for providing purified PHO polymers. Thanks are also due to Luzia Wiesli for performing protein purification and SDS-PAGE analysis. Scientific advice and the generous gift of strains and plasmids by Prof. Dr. Dieter Jendrossek (University of Stuttgart) are kindly appreciated. We thank Dr. Michael Fairhead for reading of the manuscript and Dr. Hans-Peter Fischer (ETH Zu¨rich). Financial support from the Swiss Competence Center for Materials Science and Technology (CCMX) and from an internal research grant of the Swiss Federal Institute of Materials Testing and Research (EMPA) is gratefully acknowledged. Supporting Information Available. PCR primers used for the construction of plasmids. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM9002859

Use of Extracellular Medium Chain Length Polyhydroxyalkanoate

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