EKylation: Addition of an Alternating-Charge Peptide Stabilizes

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EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins Erik J Liu, Andrew Sinclair, Andrew J Keefe, Brent L Nannenga, Brandon L Coyle, Francois Baneyx, and Shaoyi Jiang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01031 • Publication Date (Web): 25 Sep 2015 Downloaded from http://pubs.acs.org on September 26, 2015

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EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins Erik J. Liu‡, Andrew Sinclair‡, Andrew J. Keefe‡, Brent L. Nannenga, Brandon L. Coyle, François Baneyx, Shaoyi Jiang* Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, United States KEYWORDS. Biological activity • Enzymes • Peptides • Protein engineering • Protein modifications

ABSTRACT. For nearly 40 years, therapeutic proteins have been stabilized by chemical conjugation of polyethylene glycol (PEG), but recently zwitterionic materials have proven to be a more effective substitute. In this work, we demonstrate that genetic fusion of alternating-charge extensions consisting of anionic glutamic acid (E) and cationic lysine (K) is an effective strategy for protein stabilization. This bioinspired ‘EKylation’ method not only confers the stabilizing benefits of poly(zwitterions), but also allows for rapid biosynthesis of target constructs. Poly(EK) peptides of different pre-determined lengths were appended to the C-terminus of a native β-lactamase and its destabilized TEM-19 mutant. The EK-modified enzymes retained biological activity and exhibited increased stability to environmental stressors such as high temperature and high-salt solutions. This one-step strategy provides a broadly-applicable alternative to synthetic polymer conjugation that is biocompatible and degradable.

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INTRODUCTION Therapeutic proteins are now ubiquitous in medicine and account for nearly half of prescription drug expenditures in the U.S.1 Many of these beneficial proteins exhibit poor stability during production, formulation, storage, or physiological circulation. This has given rise to polymer-conjugation methods such as PEGylation, which typically improve stability at the cost of bioactivity, ease of production, biodegradability, or immunogenicity.2-4 In recent years, several alternative methods of stabilizing proteins beyond PEGylation have been proposed to address these challenges.5-7 Zwitterionic polymers such as poly(carboxybetaine) (pCB) have been used as an alternative to amphiphilic PEG and shown to impart superhydrophilic, ultra-low biofouling, and proteinstabilizing characteristics.8,

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Attachment of pCB to proteins and nanoparticles increases their

stability without hindering their functionality.5,10 In particular, pCB conjugation has been shown to improve the thermal and chemical stability of model enzyme chymotrypsin, as well as increase its substrate affinity.5 Bioinformatics studies of over 1000 polypeptides have revealed the predominance of positively charged lysine (K) and negatively charged glutamic acid (E) residues on protein surfaces at balanced ratios, suggesting an important role in protein stabilization. Additionally, repeated EK sequences (alternating or mixed) have been shown to confer non-fouling zwitterionic characteristics to surfaces and nanoparticles.11-13 This poly(EK), a natural analogue to zwitterionic pCB, is ideal for medical applications due to its biological chemistry, high biocompatibility, and enzymatic degradability.14, 15 Building on poly(zwitterionic) protein conjugation, we have developed a method to produce poly(EK)-protein constructs through biosynthesis. This ‘EKylation’ strategy allows for one-step production of recombinant conjugates with perfect structural homogeneity and eliminates the


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need for secondary chemical conjugation steps that generate products with different isoforms.16 We chose β-lactamase as a model system, as both wild-type and mutant variants of differing stabilities have been established,17 and demonstrate here that poly(EK) extensions further stabilize these proteins without drastically affecting their biological activity. Protein stabilization with rationally designed ‘amino acid polymers’ should prove valuable in alleviating the drawbacks of current conjugation methods by providing purity, molecular homogeneity, biocompatibility, and low immunogenicity.18-25

EXPERIMENTAL SECTION DNA Manipulations. E. coli strain DH10B (Life Technologies) was used for cloning. An NdeI-HindIII (New England BioLabs) DNA fragment encoding β-lactamase preceded by the OmpA signal sequence was amplified from pJG108 (Life Technologies) and ligated into the same sites of pBLN200 to place the gene under transcriptional control of the arabinose-inducible PBAD promoter.26, 27 The resulting plasmid, pBLN200-Bla was subjected to mutagenesis using the QuikChange mutagenesis kit (Agilent Technologies) to remove the stop codon. DNA cassettes encoding poly(EK) sequences 10-kDa and 30-kDa in length were commercially synthesized (GenScript USA) and fused to the 5’ end of the β-lactamase gene as HindIII-XhoI fragments. TEM-19 derivatives were obtained by introducing the G238S mutation by sitedirected mutagenesis. Protein expression and purification. Protein expression was performed using E. coli strain BL21 (DE3) (Life Technologies) cultured in Terrific Broth (TB) supplemented with kanamycin. TB was supplemented with 15% sucrose for unmodified Bla and TEM-19. Cultures were induced with 0.2% w/v arabinose at an OD600 of 0.5 and protein expression was allowed to occur


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for 4 h. Cultures were then subjected to osmotic shock, and Bla and TEM-19 variants were purified from the periplasmic fractions with an m-aminophenylboronic acid-agarose (SigmaAldrich) column as previously described28 with a modified flow rate of 0.2 mL/min. In brief, the loading and wash buffer used was 20mM triethanolamine, 500mM NaCl, pH 7 and the eluting buffer used was 500 mM borate, 500 mM NaCl, pH 7. Further purification was performed as necessary, by size exclusion chromatography (Superdex 75 10/300 column, Phenomenex) or ion exchange chromatography (HiTrap DEAE FF column, GE Healthcare). Anion exchange was carrier out using a loading buffer of 20 mM Tris, 10 mM NaCl, pH 8, with an increasing NaCl gradient. SDS-PAGE gels were imaged using a BioDoc-It Imaging System (UVP). MatrixAssisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) spectra of SEC-purified samples were obtained in linear mode using a Bruker Autoflex II. Enzyme kinetics and activity assays. Bla and TEM-19 enzymes and respective EK derivatives were diluted to 5 nM in 20 mM sodium phosphate, pH 7.0, with 10 µg/mL bovine serum albumin (BSA, Sigma-Aldrich) added as a blocking agent. Benzylpenicillin (BP, Life Technologies) solution in the same buffer was added to enzyme samples in UV-transparent 96well microplates (Corning) at varying final concentrations from 5-2000 µM. Enzyme activity (V) was measured using a microplate reader (BioTek Cytation3) as the initial linear rate of loss in substrate absorbance at 232 nm. Activity (V) vs. BP substrate concentration [S] was modeled using standard Michaelis-Menten kinetics using DataGraph 3.2 (Visual Data Tools). After determination of kinetics parameters, all further activity assays were performed at BP concentrations at least 10x Km, typically 1000 µM. All kinetics and activity experiments were performed in quadruplicate at room temperature (25°C).


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Thermal stability assays. Enzymes and conjugates were diluted to 20 nM in 20 mM sodium phosphate, pH 7.0, with 10 µg/mL BSA added as a blocking agent. Enzyme solutions were transferred to preheated polypropylene screw-top microcentrifuge tubes (Eppendorf) placed in a heating block. Temperatures ranged from 25°C to 95°C and each solution was incubated at the heightened temperature for 45 min after which they were placed on ice until being returned to room temperature immediately before activity assay as described above. Salt stability assays. Enzymes and conjugates were diluted to 20 nM in sodium phosphate buffer (20 mM, pH 7.0) or PBS with 2 M total NaCl (High-salt PBS). Temperature incubation at 80°C was performed as described above, with aliquots removed at specified times and placed on ice until assay.

Figure 1. Summary of ‘EKylation’ strategy. a) Gene for repeating amino acid polymer of lysine and glutamic acid, poly(EK), is appended to gene encoding for desired protein and the resulting construct is expressed in E. coli and purified. The result is an ‘EKylated’ fusion protein. b) Constructs generated in this study; β-lactamase (Bla) and its TEM-19 mutant were used as model proteins fused to poly(EK) peptides 10-kDa and 30-kDa in length. c) Structure of the EK peptide, showing alternating charges on each residue.


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RESULTS AND DISCUSSION We examined the effect of poly(EK) conjugation on protein stability using TEM-1 β-lactamase (Bla), a model monomeric enzyme that confers resistance to β-lactam antibiotics, as well as TEM-19, a Bla mutant with decreased thermal stability.17 Sequences coding for EK tails 10-kDa and 30-kDa in length were appended to the C-terminus of both proteins using standard molecular biology techniques. An ompA signal sequence was used to direct expression to the periplasm, which resulted in higher yields and purities than cytoplasmic expression. The resulting proteins (Fig. 1) were named EK(10k)-Bla, EK(30k)-Bla, EK(10k)-TEM19, and EK(30k)-TEM19. Following expression, purification (Fig. S1-S6), and molecular weight characterization (Fig. 2), we examined the enzymatic activity and stability to environmental stressors of all constructs. Kinetic parameters (Km and kcat) were measured using the Bla substrate benzylpenicillin, and these values are listed in Supplementary Table S1. All subsequent stability and activity studies were conducted under saturating substrate conditions. Thus, the reported changes in relative activity (V/Vmax) correspond to the fraction of enzyme remaining active following exposure to environmental stressors. Attachment of poly(EK) to the C-terminus of Bla and TEM-19 had little influence on substrate turnover numbers (kcat); Bla constructs modified with an EK(10k) tail had the same kcat as the wild type enzyme while the EK(30k)-modified protein retained 70% of the wild type value. A similar trend was observed with TEM-19 constructs, whose turnover numbers remained at about 90% and 60% of their parent upon addition of the EK(10k) and EK(30k) tails, respectively. Appending EK(30k) and EK(10k) to Bla reduced the Michaelis-Menten constant Km from 70 µM to 25 µM and 45 µM, respectively, and a similar trend (longer EK tails leading to lower Km) was observed with the TEM-19 constructs (Table S1). This indicates that the addition of EK extensions leads to an apparent increase in substrate affinity, an effect that we


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previously observed in poly(zwitterionic) enzyme-pCB conjugates, which can be explained by superhydrophilic properties of poly(zwitterionic) materials promoting specific hydrophobic interactions required for substrate binding compared to PEG’s amphiphilic characteristics.5 Fusion of poly(EK) extensions may similarly improve the affinity of Bla for benzylpenicillin.

Figure 2. Purification and characterization of EKylated constructs. a) Representative SDSPAGE gels demonstrating the expression and purification of Bla, EK(30k)-Bla, and EK(10k)-Bla showing whole cell (WC), cytosolic (Cyto), and periplasmic (Peri) fractions and products after agarose (BA) and size-exclusion (SEC) purifications. The desired products are highlighted. b) MALDI-TOF mass spectra of Bla, EK(10k)-Bla, and EK(30k)-Bla constructs.


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Like chemical conjugation of pCB, poly(EK) tails conferred increased thermostability to Bla and TEM-19 (Fig. 3).5 The responses to thermal stress highlighted the importance of poly(EK) tail length—addition of the short EK(10k) extension increased the transition midpoint temperature by approximately 15°C without affecting the shape of the unfolding curve, while the longer EK(30k) extension stabilized the enzymes at very high temperatures.

Figure 3. Relative thermal stability of Bla conjugates (top) and TEM19 conjugates (bottom). For both enzymes, the EK(10k) modification confers a similar stability benefit throughout, while the EK(30k) modification increases stability most in more extreme conditions above Tm. Curves are drawn to guide the eye.


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As seen in Fig. 3, EK(30k) increased the midpoint transition temperature by approximately 2030°C and led to a modified thermal unfolding profile. These results indicate that there is a slight tradeoff between enzymatic activity at lower temperatures and stability at extreme temperatures, and suggest that different lengths of poly(EK) extensions may be optimal for different desired applications (e.g., catalytic or storage conditions). Remarkably, proteins modified with EK(30k) retained 30-40% of their initial activity after 1 h incubation at 95°C, a 200-300% improvement over the wild-type enzyme, or a 150-200% improvement in absolute activity. We also observed poly(EK) to confer notable protection from activity loss when thermal stability experiments were conducted in the presence of high salt (NaCl) concentrations (Fig. 4). Indeed, while Bla and TEM-19 modified with EK(30k) only experienced a small (~20%) loss of activity after 90 min of incubation at 80°C in the presence of 2 M NaCl, unmodified enzymes were nearly completely inactivated. A more pronounced stabilizing effect of EK extensions in high-salt conditions suggests that the E and K residues interact with counterions in solution to buffer deleterious ionic interactions experienced by the protein at high temperatures and prevent its destabilization.29 Similar studies were conducted without the use of a blocking agent (serum albumin) in the assay buffer. This resulted in a much more rapid loss of enzymatic activity by unmodified enzymes (Fig. S8), suggesting that like non-fouling EK peptides, poly(EK) extensions reduce interactions with other proteins and surfaces that lead to denaturation. Thus, poly(EK) tails may act as a general protein stabilizer by preventing physical and chemical stressors from interfering with protein structure and activity.


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Figure 4. Relative temperature stability of Bla and EK(30k)-Bla (top), as well as TEM19 and EK(30k)-TEM19 (bottom), as activity retention after incubation in high salt (2M, solid lines and points) and normal salt (20mM, dotted lines and open points) conditions at 80°C. Similar substantial activity loss is seen for unmodified proteins in both conditions, while proteins containing poly(EK) retain high activity, especially in high salt. Curves are drawn to guide the eye.


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CONCLUSIONS In summary, we have demonstrated a new strategy for protein stabilization that relies on fusing poly(EK) extensions to their termini via genetic engineering. The tails were developed as a peptide analogue of the zwitterionic polymer pCB that exhibits similar low-fouling and biocompatible properties. We successfully appended poly(EK) tails of well-defined lengths to the C-terminus of the model enzyme β-lactamase and showed that these constructs retained biological activity but exhibited increased protein stability, consistent with previous results with zwitterionic pCB polymers and poly(EK) peptides. Poly(EK) extensions of different lengths led to different protecting effects, with the shorter poly(EK) chain conferring additional stability without impacting bioactivity, and the longer poly(EK) chain imparting a small loss in activity but a significant increase in thermostability. Poly(EK) extensions were also found to reduce the Km, thereby improving protein-substrate affinity. Such marked stability improvements conferred through the use of a biologically-designed and well-defined method of poly(EK) addition should find applications in fields ranging from drug delivery to protein therapeutics. In addition to N- or C-terminal fusions, poly(EK) tails will confer a stabilization advantage when conjugated to proteins in a site-specific manner through non-canonical amino acids or other strategies.

ASSOCIATED CONTENT Supporting Information. SDS-PAGE gels detailing the expression and purification of the proteins, SEC curves, additional stability data, and kinetics parameters are included in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by Army Research Office (63926-LS-II), Office of Naval Research (N000141010600) and National Science Foundation (1264477) REFERENCES (1)

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TABLE OF CONTENTS GRAPHIC

Title: EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins

Authors: Erik J. Liu‡, Andrew Sinclair‡, Andrew J. Keefe‡, Brent L. Nannenga, Brandon L. Coyle, François Baneyx, Shaoyi Jiang*


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