In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films.

polyurethane catheters via eSrtA stripping reaction. (A) Representative merged fluorescent and bright field microscopy images obtained from polyurethane catheters modified with pentaglycine motifs and reacted with biotin-LPETG and eSrtA. Biotin-LPETG was then stripped from catheters using various concentrations of GGG peptide and eSrtA. Catheters were incubated with Cy3-labeled streptavidin at 0.1 mg . mL -1 for 30 min to assess the surface density of biotin. (B) Fluorescence intensity was measured using Image J and expressed as mean ± standard deviation for three individual catheter segments for each reaction condition (n=3). The concentrations of GGG peptide and eSrtA, which were used to strip biotin-LPETG from catheters are summarized in (C). images of polyurethane catheters modified with pentaglycine motifs and reacted with biotin-LPETG and eSrtA. Biotin-LPETG was then stripped from catheters using various concentrations of GGG peptide and eSrtA. Catheters were incubated with Cy3-labeled streptavidin at 0.1 mg . mL -1 for 30 min to assess the surface density of biotin. (B) Fluorescence intensity was measured using Image J and expressed as mean ± standard deviation for three individual catheter segments for each reaction condition (n=3). The concentrations of GGG peptide and eSrtA, which were used to strip biotin-LPETG from catheter surfaces are summarized in a Table of reaction conditions (C).  Red-labeled TM LPETG (TM catheter) and initially deployed in the rat jugular vein for 7 days. Triglycine with or without eSrtA was then delivered intravenously via the dorsal penile vein and 1 h later catheters were explanted and imaged. To examine in situ recharging, TM catheters were stripped in vivo after being deployed in the rat jugular vein for 7 days, as described above, and 24 h later, Texas Red-TM LPETG along with eSrtA was administered intravenously via the dorsal penile vein. Catheters were explanted 1 h later for imaging (n=5).  Mutations from the native protein sequence are colored red. OmpA tag sequence is highlighted in blue, which facilitates transport of TM LPETG to the periplasmic space of E. coli to optimize folding and is cleaved in final mature TM LPETG . The FLAG peptide sequence of DYKDDDDK at the N-terminus is introduced for protein purification. It has been previously demonstrated that the conversion of the internal methionine residue to leucine maximizes the stability of TM 70 . A second mutation was made to convert the internal RH sequence, which is an active trypsin cleavage site to GQ. The additional sequence at the C-terminus includes a GGGGSGGGGS spacer and the LPETG sortase recognition motif.

Supplementary
An extra glycine was added at the C-terminus as this was previously demonstrated to maximize sortase activity.

Supplementary Methods
Expression of TM LPETG . The minimal fragment of human thrombomodulin, epidermal growth factor-like domains 4, 5 and 6 (TM456), was cloned into the Sigma pFLAG ATS expression vector.
The sequence is provided in Supplementary Table 1. Following transformation of BL21 cells, a fresh LB agar plate was streaked and a single cell colony was then inoculated into 50 mL of media supplemented with 0.4 % glucose and 50 μg . mL -1 ampicillin and cultured for 16 h at 37°C and 225 RPM.
A total of 25 mL of fully grown starter culture (OD600 = 1.20) was then added to 500 mL of media supplemented with 0.4 % glucose and 50 μg . mL -1 ampicillin, and cultured at 37°C and 225 RPM. Upon cell growth to OD600 = 0.9, IPTG was then added at a final concentration of 1 mM to induce TM LPETG expression and the culture incubated for an additional 4 h at 37°C and 225 RPM. Cell cultures were centrifuged at 4,000x RCF at 4°C for 10 min and stored at 4°C. A standard osmotic shock protocol was performed on stored cell pellets to extract the crude periplasmic proteins. Cell pellets were first warmed to room temperature and re-suspended in 40 mL . g -1 cells of 0.5 M sucrose, 0.03 M Tris-HCl (pH 8.0).
Suspended cells were evenly distributed into round bottom centrifuge tubes (60 mL per tube). EDTA was then added at a final concentration of 1 mM and cells incubated with gentle shaking for 10 min at room temperature. The cell suspension was centrifuged at 3,500x g for 10 min at 10°C and the supernatant decanted. The cell pellet was rapidly resuspended in 25 mL of ice-cold, distilled water per gm of cell pellet for 10 min and the cell suspension centrifuged at 3,500x RCF for an additional 10 min at 4°C. A total of 35 mL of supernatant was removed from each tube and transferred to clean round bottom centrifuge tubes. These were clarified by further centrifugation at 25,000x RCF for 25 min at 4°C and sterilized using a 0.22 µm filtration system. Anti-FLAG immunoaffinity chromatography (Sigma) was performed on the clarified supernatant per manufacturer's instructions. SDS-PAGE analysis was conducted and total protein quantification was performed using a standard Bradford assay.
Bacterial expression of evolved sortase and wild-type sortase. E. coli BL21 transformed with pET29 wild-type sortase or evolved sortase expression plasmids (sequence provided in Supplementary   Table 1) were cultured at 37°C and 225 RPM in LB media supplemented with 50 μg . mL -1 kanamycin.
Upon OD600 = 0.8, IPTG was added to a final concentration of 0.4 mM and protein expression was induced for 3 h at 30°C. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgCl 2 , 2 units . mL -1 DNAseI (NEB), 260 nM aprotinin, 1.2 μM leupeptin, and 1 mM PMSF). Cells were lysed by sonication on ice and the clarified supernatant was purified by column chromatography using Cobalt Talon Resin (Clontech Laboratories) following the manufacturer's instructions. Fractions that were >95 % purity, as judged by SDS-PAGE, were consolidated and dialyzed against Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl) using PD-10 columns (GE Healthcare) and stored as 5 mg . mL -1 stocks at 4°C.