PROLinkâSingle Step Circularization and Purification Procedure for

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PROLink - A single step circularization and purification procedure for the generation of an improved variant of human growth hormone Nicolas Rasche, Jason Tonillo, Marcel Rieker, Stefan Becker, Brent Dorr, Dmitry Ter-Ovanesyan, Ulrich A.K. Betz, Bjoern Hock, and Harald Kolmar Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00137 • Publication Date (Web): 23 Apr 2016 Downloaded from http://pubs.acs.org on April 23, 2016

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Bioconjugate Chemistry

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PROLink - A single step circularization and purification procedure for the

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generation of an improved variant of human growth hormone

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Nicolas Rasche†,‡, Jason Tonillo‡, Marcel Rieker†,‡, Stefan Becker‡, Brent Dorr║, Dmitry Ter-

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Ovanesyan§, Ulrich A.K. Betz‡, Björn Hock‡, Harald Kolmar†,*

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†

Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt

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‡

Merck KGaA, Frankfurterstr. 250, 64293 Darmstadt, Germany

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║

present address: GlaxoSmithKline, Platform Technology & Science, King of Prussia, PA 19406, United States

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§

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and Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, United States

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ABSTRACT

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Human growth hormone (hGH) plays an important role during human development and is

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also an approved therapeutic for the treatment of several diseases. However, one major

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drawback of hGH is its short circulating half-life requiring frequent administrations which is

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inconvenient and painful for the patients. Recent publications indicate that circularization

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greatly increases the stability of proteins due to their protection from exoproteolytic attack

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and a higher thermal stability of the circular form. Using sortase A, a transpeptidase isolated

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from Staphylococcus aureus, we developed a single step solid phase circularization and

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purification procedure resulting in a circular version of hGH with improved properties. We

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could show that circular hGH binds to the recombinant hGH receptor with binding kinetics

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similar to those of linear hGH and that circularization does not alter the biological activity of

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hGH in vitro. Besides, circular hGH showed almost complete resistance towards

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exoproteolytic attack and slightly increased thermal stability which could possibly translate

Department of Molecular and Cellular Biology, Harvard University, Cambridge MA 02138

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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into an extended plasma half-life. The single step solid phase circularization and purification

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procedure is in principle a generic process, which could also be applied for other proteins that

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meet the requirements for circularization.

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INTRODUCTION

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Human growth hormone (hGH) is secreted by somatotropic cells of the anterior pituitary

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gland and plays an important role in human development and adolescence. In the late 1950s

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hGH isolated from human cadaveric pituitaries was used to treat patients suffering from

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growth hormone deficiency (GHD).1 However, it was only with the advent of recombinant

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DNA technology that the production of hGH at an industrial scale became possible. Since

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then the application of hGH has been approved for several indications including growth

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disorders in children, GHD in adults, HIV-related wasting, Turner syndrome, Prader-Willi

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syndrome, chronic renal insufficiency and others.2 Although hGH therapy has been applied

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successfully for decades, it still retains some critical flaws. One of the major drawbacks is the

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short plasma half-life of hGH which is about 3.4 h if administered subcutaneously (SC) and

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0.36 h if administered intravenously (IV).3 Due to the short circulating half-life daily or

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thrice-weekly administration is required which is inconvenient and painful for the patients.4

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To overcome this, great efforts have been made to develop long-acting hGH preparations like

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zinc complexes or microspheres. However, these approaches suffer from other disadvantages

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such as low loading efficiencies of microspheres, high initial burst release, protein

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aggregation, hydrophobic microsphere surfaces etc.5 Another widely used technique to

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increase the plasma half-life of hGH is PEGylation.6 In case of PHA-794428 PEGylation of

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hGH resulted in a 10- to 20-fold increase of its in vivo half-life.3 Unfortunately, a clinical

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Phase II trial revealed the occurrence of injection-site lipoatrophy in GHD patients treated

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with PEGylated hGH, which ultimately led to termination of the trial.7 In an alternative

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approach, a fusion protein was designed by genetically fusing conformationally disordered


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polypeptide chains to the N- and/or the C-termini of the native hGH sequence.8,9 Although in

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vitro potency of VRS-317 carrying terminal XTEN extension polypeptides was reduced 12-

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fold, in vivo efficacy was increased due to prolonged stability in tissues and organs.8

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Sortase A (SrtA) belongs to a group of prokaryotic enzymes that covalently attach proteins to

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the bacterial cell wall.10 Due to its high specificity SrtA gains an increasing interest in

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biotechnological applications such as site specific protein labeling, the production of fusion or

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cyclic proteins and protein purification.11-13 SrtA is a Ca2+ dependent thiol containing

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transpeptidase that recognizes an LPXTG motif and cleaves the peptide bond between the

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threonine and glycine residues with concomitant formation of a thioester intermediate. The

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thioester is resolved by a nucleophilic attack of the N-terminal amino group of an oligo-

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glycine peptide, resulting in the formation of a new amide bond. SrtA was successfully used

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to cyclize disulfide-rich peptides and miniproteins, including members of the cyclotide and

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conotoxin family.14,15 In 2011 Popp et al.16 showed that cytokines with a four-helix bundle

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structure can be circularized using SrtA. Circularization of PEGylated interferon alpha 2a

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(INFα2a) increased its circulatory half-life in mice from about 6 h for the PEGylated linear

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INFα2a to 31 h for the circular version. The gain in stability due to circularization was

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attributed to a higher thermal stability of the circular protein and an increased resistance

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towards exopeptidases, which are responsible for protein degradation and involved in the

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regulation of natural protein turnover. Prerequisites for an efficient circularization via SrtA

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are a good accessibility and close proximity of N- and C-termini and engineering of an N-

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terminal glycine and C-terminal LPXTG motif. The crystal structure of hGH suggests that its

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termini are in such a favorable orientation, hence the production of a circular hGH with

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improved stability might be possible.17 The choice to use SrtA in our circularization

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procedure additionally opens the opportunity to combine circularization with purification as

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described in case of sortase mediated site-specific bioconjugation18 and one-step purification



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of recombinant proteins13, thereby implementing a highly effective and scalable production

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procedure for circular hGH.

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Here we present a single step purification and circularization procedure resulting in circular

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hGH. We show that circularization of hGH results in an increased resistance towards

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exoproteolytic attack but does neither impair its binding to the hGH-receptor in vitro nor

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affect its stimulatory effect on Nb2 rat lymphoma cells. The single step purification and

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circularization procedure described here is also applicable for other proteins that meet the

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requirements for circularization.

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RESULTS AND DISCUSSION

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Circularization of hGH. Recent studies demonstrated that circularization of proteins via

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SrtA mediated transpeptidation is possible and often results in increased thermal stability and

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plasma half-life.12,16 However, certain structural requirements need to be met which determine

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the efficiency of the circularization reaction. Most importantly, the termini of the protein to be

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circularized should be in close proximity and show a certain degree of flexibility. The crystal

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structure of hGH (Figure 1a) allows the conclusion that in case of hGH these requirements are

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met. Only the disulfide bond located at the very C-terminus of hGH might decrease flexibility

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and thereby potentially hinder an efficient circularization. To assess if circular hGH can be

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obtained in a SrtA catalyzed reaction, a modified version of hGH was expressed in HEK293

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cells. An LPETG motif was genetically fused to the C-terminus of hGH followed by a

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hexahistidine tag for ease of purification. Additionally, a leader sequence was chosen which,

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after processing and export, yields a mature hGH with two glycine residues at its N-terminus.

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After transient transfection and expression recombinant hGH was purified via IMAC and

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subsequently transferred to SrtA reaction buffer. Purified hGH was then applied to a typical

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SrtA reaction resulting in successful protein circularization (Figure 1b). However, in addition

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to circular monomeric hGH, the SrtA reaction also resulted in dimeric and multimeric hGH - 4 - Environment ACS Paragon Plus

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forms. To isolate the monomeric variant of circular hGH, the SrtA reaction mixture was

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passed through an IMAC column and the flow through was collected. In this way SrtA and

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linear hGH substrate as well as linear hGH oligomers, all carrying a hexahistidine tag were

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retained on the column while the circular products, which lacked a his-tag as a result of SrtA

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processing, were found in the flow through (Figure S1). Circular hGH monomer could then be

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separated from circular dimer and circular multimers via size exclusion chromatography

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(Figure 1c) and successful cyclization was confirmed via mass spectrometry (Figure 1d,e).

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Figure 1. Circularization and characterization of hGH. (a) Crystal structure of hGH (PDB code 3HHR). Disulfides are shown in yellow, N-terminus in orange and C-terminus in teal. (b) SDS-PAGE analysis of a SrtA reaction with hGH: 1 SrtA, 2 hGH, 3 SrtA reaction 0 min, - 5 - Environment ACS Paragon Plus


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4 SrtA reaction 4h. (c) SDS-PAGE analysis of purified linear 1 and circular monomeric hGH 2. (d) MS characterization of purified circular monomeric hGH, intact mass 22663 (calculated 22665). (e) MS/MS identification of a tryptic peptide comprising the hGH C-terminus with the SrtA motif fused to the N-terminal glycine.

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Receptor binding and biological activity. To verify that circularization of hGH does not

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impair its affinity to the hGH receptor bio-layer interferometry (BLI) measurements were

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performed. A recombinant hGH receptor Fc chimera (R&D systems, 1210-GR-050), was

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immobilized on ForteBio biosensors and binding of soluble hGH to the receptor was

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monitored using a ForteBio Octet RED96 (Figure S2). Both, the linear as well as the circular

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form of hGH showed similar binding kinetics with dissociation constants of 0.22 nM and

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0.18 nM, respectively (Table 1). These findings are not unexpected since the co-crystal

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structure of hGH together with the extracellular domain of its receptor17 indicates that N- and

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C-terminus of hGH reach into the solvent and are not involved in receptor binding. Therefore,

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it can be concluded that circularization of hGH does not impair its binding to the hGH

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receptor in vitro.

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Table 1. Melting points and binding characteristics of linear and circular hGH against recombinant hGH receptor (determined by DSF and BLI measurements, respectively). KD (nM)

kon (105 M-1s-1)

kdis (10-4s-1)

Tm (°C)

linear hGH

0.22 ± 0.005

6.65 ± 0.04

1.44 ± 0.04

71.8

circular hGH

0.18 ± 0.004

7.88 ± 0.04

1.39 ± 0.03

73.9

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To demonstrate that the biological activity of hGH is unaffected by the circularization

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procedure a proliferation assay with Nb2-11 rat lymphoma cells was performed.19 Nb2-11

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cells are of the pre-T cell origin and their proliferation is dependent on hGH or mammalian

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lactogens like prolactin. After starvation in Fischer´s medium without fetal bovine serum

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(FBS) the cells were treated with different concentrations of either linear or circular hGH. - 6 - Environment ACS Paragon Plus

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After 3 days of incubation, the growth-promoting activity of the two different hGH variants

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was evaluated in a proliferation assay. Cells treated with 10% FBS served as positive control

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while cells treated with phosphate buffered saline (PBS) were used as negative control. The

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obtained proliferation curve (Figure 2) indicates strong stimulatory effects for both hGH

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variants. EC50 values of 25 pg/ml and 28 pg/ml for the linear and the circular hGH variant,

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respectively, suggest that the stimulating activities of both proteins are very similar, leading to

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the conclusion that circularization also does not impair the biological activity of hGH in vitro.

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Figure 2. Proliferation stimulating effects of linear and circular hGH on Nb2-11 rat lymphoma cells. Viability of Nb2-11 cells was measured after incubation with linear hGH (blue) or circular hGH (orange). Level of viability without addition of hGH is indicated as dashed line, buffer (red) or 10% FCS (green).

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Stability of circular hGH. It has been shown that circularization can lead to an increased

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stability, potency and bioavailability of proteins and peptides.14,20-23 These effects are mainly

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attributed to an improved thermal stability and to protection of cyclic proteins from

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exoproteolytic degradation.24,25 To assess if also circularization of hGH comes along with

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these benefits, thermal stability and resistance to exoproteolytic degradation of linear and - 7 - Environment ACS Paragon Plus


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circular hGH were compared. First, differential scanning fluorimetry (DSF) was performed to

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evaluate the impact of circularization on the thermal stability of hGH. Via DSF thermal

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denaturation of proteins can be monitored through binding of the fluorescent dye SYPRO

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orange26. Figure S3 shows the first derivation of the melting curve obtained from DSF

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measurements of linear and circular hGH. Both hGH variants exhibit relatively high melting

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points. However, with 73.9 °C the melting point of circular hGH was only slightly higher than

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for the linear variant with 71.8 °C (Table 1). Hence, the impact of circularization on thermal

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stability is rather subtle in this case.

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To assess if circularization protects hGH from exoproteolytic attack the resistance of linear

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and circular hGH against exoproteolytic degradation was compared. Both hGH variants were

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incubated with carboxy peptidase Y (CPY) as well as with aminopeptidase N (APN) at 37° C

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for 45 min and degradation products were analyzed via SDS PAGE (Figure 3) and mass

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spectrometry. Treatment of linear hGH with exopeptidases resulted in almost complete

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proteolysis of the full length protein and a characteristic pattern of degradation products

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(Figure 3, compare lanes 1-3). In contrast, incubation of circular hGH with APN and CPY

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only led to a slight smear of hGH fragments in the gel while the main part of the protein

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remained intact (Figure 3, compare lanes 4-6). Interestingly, the migration behavior of cyclic

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hGH in SDS PAGE changed upon incubation with CPY. The circular untreated hGH runs at

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approximately 20 kDa while the exopeptidase treated hGH displays a delayed migration

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behavior. However, mass spectrometry analyses of the shifted species confirmed that it is still

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the intact cyclic hGH (Figure S4) which leaves this phenomenon unresolved at the moment.

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Nevertheless, some degree of degradation was also observed for circular hGH resulting in a

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slight smear in SDS-PAGE (Figure 3b, lanes 5,6) which might originate from proteinase A

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which was reported to be a common contamination of CPY isolated from baker´s yeast.27


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Figure 3. Digestion of linear and circular hGH with aminopeptidase N (APN) and carboxypeptidase Y (CYP). Linear hGH (1) was either incubated with CPY (2) or with CPY and APN (3). Circular hGH (4) was either incubated with CPY (5) or with CPY and APN (6).

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Single step circularization and purification. Circularization of hGH via SrtA results in

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a heterogeneous mixture of substrate, different products and SrtA, thereby requiring complex

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purification steps reducing the final yield of cyclic hGH. To increase the overall yield and to

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overcome the need for extensive purification procedures we established a single step

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circularization and purification procedure similar to a strategy described by Mao et al.13 and

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Warden-Rothman et al.28 To this end, hGH was expressed as N-terminal SrtA fusion protein

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with a C-terminal hexahistidine tag which allowed purification and circularization to be

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performed in a single step (Figure 4a). Since expression in HEK293 cells took several days

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SrtA had already processed the majority of the fusion protein before a purification was

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possible (data not shown). Matsunaga et al. used the Ca2+ chelator BAPTA to overcome this

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problem in their cell-free expression system.29 Unfortunately, adding Ca2+ chelating agents

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such as EGTA to our cell culture did not result in higher yields of fusion protein. One

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possibility to overcome this problem could be the generation of a switchable SrtA variant.

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Hirakawa et al. for example engineered a variant of SrtA which is independent of Ca2+ ions.30

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In contrast to that, the generation of a SrtA which is strictly dependent on higher - 9 - Environment ACS Paragon Plus


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concentrations of Ca2+ might also be possible using e.g. established yeast display systems for

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sortase A optimization.31,32 For now we solved this issue using E. coli as alternative

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expression host since it was shown previously that highly efficient production of soluble

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recombinant hGH is possible.33 The additional reduction of expression time to 3h facilitated

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isolation of fusion proteins with only little formation of prematurely processed products.

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Following cell disruption, the E. coli cell extract was loaded onto a Ni2+-affinity column

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where the hGH-SrtA fusion protein bound to the affinity matrix and unbound material was

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washed away. Subsequently, the SrtA reaction was initiated by an on column buffer exchange

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from running buffer to SrtA reaction buffer. The column was then removed from the LC

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system and incubated at 37°C for 4h to enable an efficient circularization. Subsequently, the

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column was reinserted into the LC system and the flow trough was collected. SDS-PAGE and

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mass spectrometry analysis confirmed efficient formation of cyclic monomeric hGH while

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only a small amount of the substrate fusion protein could be detected (Figure 4b, lane 2). In

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this way purification and circularization could be combined in a single step and also

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diminished the formation of hGH dimers and multimers. After on-column circularization the

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column was washed with elution buffer containing 250 mM Imidazole and the eluate was

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analyzed via SDS-PAGE (Figure 4b, lane 3). As expected, only very little of the intact hGH-

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SrtA fusion protein remained on the column while the majority of bound protein was indeed

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the processed SrtA. Hence, very efficient on-column circularization could be confirmed.

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Figure 4. Single step circularization and purification. a) Scheme of on-column circularization procedure. Purple: affinity tag, yellow: SrtA, teal: LPETG-motif, orange: oligo-glycine motif, blue: protein of interest (POI). First, fusion protein binds to the column via affinity tag then SrtA is activated, resulting in elution of the circular POI while SrtA remains on column. b) SDS-PAGE analysis after on column circularization. 1) E. coli cell extract, 2) protein eluted after SrtA activation, 3) remainder of protein on column, eluted with imidazole containing buffer.

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CONCLUSION

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Backbone cyclization of peptides and proteins has been shown to come along with benefits

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such as improved biological activity and increased in vivo stability. In case of the four-helix

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bundle type protein INFα2 a dramatic increase of its in vivo half-life was achieved by efficient

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circularization using SrtA.16 Hence, it was suggested that circularization via SrtA might also

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be well suited for similar types of proteins fulfilling certain structural requirements such as

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flexibility and close proximity of N- and C-terminus. hGH, although approved for several

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therapeutic indications since decades now, still suffers from its short circulating half-life but,

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looking at its crystal structure (Figure 1a), it seemed to be predestined for circularization.

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Both of its termini appear to be rather flexible and are not involved in hGH receptor binding.17

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In the present work, we successfully applied SrtA to generate a cyclic form of hGH. We could

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demonstrate that compared to linear hGH circular hGH indeed showed a significantly higher

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resistance towards exoproteolytic attack from carboxypeptidase Y and aminopeptidase N 11 - Environment ACS Paragon -Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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while the biological activity, thermal stability and affinity to the hGH receptor of both

246

proteins were very similar. Moreover, a single-step circularization procedure was established

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that relies on the expression in E. coli of a SrtA-hGH fusion protein containing a C-terminal

248

oligohistidine extension. Since sortase A activity is Ca2+ dependent, the fusion protein can be

249

purified via capturing to a metal chelate column in the absence of calcium ions. Addition of

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Ca2+ results in the release circularized hGH in good yields which drastically simplifies the

251

purification procedure.

252

Upon Sortase A-mediated circularisation of GG-hGH-LPETG we observed a significant

253

fraction of circular hGH dimers. hGH contains two distinct receptor binding sites (site 1 with

254

high affinity; site 2 with low affinity) that each contribute to the formation of a functional

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receptor dimer, thereby activating signal transduction.17,34,35 It remains to be elucidated

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whether these artificial circular hGH dimers, which contain two high as well as two low

257

affinity binding sites, display biological activity and in vitro stability comparable to their

258

monomeric circular counterparts.

259 260

EXPERIMENTAL SECTION

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hGH circularization via SrtA. SrtA reactions were essentially performed as described by

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Popp et al.36. For cyclization in solution, 80 µM substrate and 50 µM SrtA were incubated for

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4h at 37 °C in 150 mM NaCl, 50 mM Tris-HCl pH 7.5 and 5 mM CaCl2. Subsequently,

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reactions were passed through a 1 ml HisTrap HP column (GE Healthcare) and the flow

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through was collected and concentrated using Amicon Ultra-15 centrifugal filter units (EMD

266

Millipore). The cyclic monomeric form of hGH was isolated from the concentrated sample

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via HiLoad 16/600 Superdex 75 column (GE Healthcare) at a flow rate of 1 ml/min in 150

268

mM NaCl, 50 mM Tris-HCl pH 7.5. For on-column circularization, E. coli extract containing

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hGH-SrtA fusion protein was passed through a 1 ml HisTrap HP column. Afterwards, the

270

column was washed with 10 ml of 500 mM NaCl, 50 mM Tris-HCl pH 7.5, 15 mM imidazole 12 - Environment ACS Paragon -Plus

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and then equilibrated with 10 ml of 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM CaCl2.

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After equilibration with Ca2+ containing buffer, the column was sealed and incubated at 37°C

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for 4h. Subsequently, the flow-through containing circular hGH was collected and analyzed

274

via SDS-PAGE. The remainder of protein bound to the column was eluted with 500 mM

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NaCl, 50 mM Tris-HCl pH 7.5, 250 mM imidazole.

276

Biolayer Interferometry. BLI experiments were conducted using the Octet RED system

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(ForteBio). Anti-human Fc capture (AHC) biosensors (ForteBio) were equilibrated in PBS for

278

20 min followed by loading with recombinant human growth hormone receptor Fc chimera

279

(R&D Systems). Baseline was obtained by incubation in kinetics buffer (0.1% BSA, 0.02 %

280

TweenTM-20 in PBS pH 7.4) for 300 s. Association and dissociation were performed for 600 s

281

in kinetics buffer, respectively. All steps were performed in 200 µl kinetics buffer at 25 °C

282

and 800 rpm sensor agitation. Sensograms were fitted using Langmuir binding model.

283

Mass spectrometry. LC/MS analysis was performed using an Acquity UPLC System

284

(Waters) coupled to a Synapt-G2 mass spectrometer (Waters). The protein solution was

285

diluted with 0.1 % TFA to 1 mg/ml and 1 µl was trapped on a desalting column (Waters part

286

no. 186004032). After 2 min washing with 1 % Buffer A (0.1 % TFA) and 20 µl/min flow

287

rate, the protein was eluted with a gradient from 5 % to 90 % Buffer B (acetonitrile, 0.1 %

288

TFA) within 7 min. Data were acquired in resolution mode with positive polarity and in a

289

mass range from 400 to 6,000 m/z. Additional instrument settings were set as follows:

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capillary voltage 3 kV, sampling cone to 34 V, extraction cone to 4.1 V, source temperature

291

80 °C, desolvation temperature 150 °C, cone gas 10 l/h, and desolvation gas 400 l/h. Spectra

292

were deconvoluted with the MaxEnt1 algorithm within the MassLynx software.

293

For the preparation of MALDI matrix α-Cyano-4 hydroxy-cinamic acid was dissolved in

294

ethanol (saturated solution) and 10 µl of the solution were diluted with 90 µl 30 % ACN,

295

0,1% TFA. 1 µl trypsin digested protein (1 mg/ml) were mixed with 1 µl MALDI matrix,

296

spotted on a MTP AnchorChip 600/384 (Bruker) und dried at room temperature. The spot was 13 - Environment ACS Paragon -Plus


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washed once with 0.1 % TFA und peptides were analyzed with a Bruker Ultraflex MALDI

298

TOF/TOF mass spectrometer under control of FlexControl 3.4. The peptide of interest was

299

fragmented with laser induced dissociation and acquired spectra were processed with

300

FlexAnalysis 3.4 and BioTools 3.2.

301

Proliferation assays. To compare the biological activity of linear and circular hGH a

302

proliferation assay with Nb2-11 rat lymphoblasts was performed

303

cultured in Fischer´s medium supplemented with 10% fetal bovine serum (FBS), 0.075%

304

sodium bicarbonate, 0.05 mM 2-mercaptoethanol (2ME) and 2 mM glutamine. For the

305

proliferation assay 4x104 cells/well in Fischer´s medium without FBS were seeded into 96

306

well cell culture plates and starved for 24h before addition of hGH or FBS. After incubation

307

for 3 days cell density was determined using CellTiter 96® AQueous One Solution Cell

308

Proliferation Assay according to the manufacturer’s instructions.

309

Differential scanning fluorimetry. Melting temperatures were determined in triplicates

310

using StepOnePlusTM Real-time PCR instrument (Life Technologies). 25 µg protein were

311

added to 50 µl 1x DPBS, 30 x Sypro Orange (Invitrogen) and transferred into MicroAmp®

312

Fast 8 Tube Strips (Applied Biosystems). After spinning, the mixture was put on ice until

313

analysis. The samples were heated continuously from 25 to 99 °C at a heating rate of

314

1 °C/min. Data was analyzed via StepOnePlusTM v. 2.2.2 software.

315

Exopeptidase assays. Protection of hGH variants from exoproteolytic degradation was

316

assessed by in vitro protease assays using carboxypeptidase Y from S. cerevisiae (Sigma,

317

product code: C3888) and aminopeptidase N (Merck Millipore, 164605). 10 µg of linear or

318

circular hGH were incubated with 2 µg of CPY and/or 2 µg of APN at 37°C for 45 min in 60

319

µl 150 mM NaCl, 50 mM Tris-HCl pH 7.5. Reactions were subsequently analyzed via SDS-

320

PAGE and mass spectrometry.

321


19,37,38

. Nb2-11 cells were

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322

ACKNOWLEDGEMENTS

323

This work was funded by Merck KGaA Darmstadt in the frame of the Merck Healthcare

324

Innovation Cup. We specially thank Brent Dorr, Emily Fang, Stefan Luzi, Dmitry Ter-

325

Ovanesyan, Vanessa Burns and Siegfried Neumann for their great work during concept

326

generation of the project.

327

328

Supporting Information. Supplementary figures documenting the purification of hGH, its

329

receptor binding kinetics, thermal and proteolytic stability (PDF).

330

331

AUTHOR INFORMATION

332

Corresponding Author:

333

*E-mail: [email protected]

334 335

Notes

336

The authors declare no competing financial interest.



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Figure 2. Proliferation stimulating effects of linear and circular hGH on Nb2-11 rat lymphoma cells. Viability of Nb2-11 cells was measured after incubation with linear hGH (blue) or circular hGH (orange). Level of viability without addition of hGH is indicated as dashed line, buffer (red) or 10% FCS (green). 80x41mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 3. Digestion of linear and circular hGH with aminopeptidase N (APN) and carboxypeptidase Y (CYP). Linear hGH (1) was either incubated with CPY (2) or with CPY and APN (3). Circular hGH (4) was either incubated with CPY (5) or with CPY and APN (6). 81x56mm (300 x 300 DPI)


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Figure 4. Single step circularization and purification. a) Scheme of on-column circularization procedure. Purple: affinity tag, yellow: SrtA, teal: LPETG-motif, orange: oligo-glycine motif, blue: protein of interest (POI). First, fusion protein binds to the column via affinity tag then SrtA is activated, resulting in elution of the circular POI while SrtA remains on column. b) SDS-PAGE analysis after on column circularization. 1) E. coli cell extract, 2) protein eluted after SrtA activation, 3) remainder of protein on column, eluted with imidazole containing buffer. 150x76mm (300 x 300 DPI)



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61x23mm (300 x 300 DPI)


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