Experimental - Profiling Sortase Substrate Specificity using Peptide Libraries

5.1 Protein Expression and Purification

Sequences of proteins used in this study:

SrtAStaph (initiator methionine absent in purified protein)

GSSHHHHHHSSGLVPRGSHMQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPE QLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETR KYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK

SrtAAnth (initiator methionine absent in purified protein)

GSSHHHHHHSSGLVPRGSHMDASKIDQPDLAEVANASLDKKQVIGRISIPSVSLEL PVLKSSTEKNLLSGAATVKENQVMGKGNYALAGHNMSKKGVLFSDIASLKKGDKIY LYDNENEYEYAVTGVSEVTPDKWEVVEDHGKDEITLITCVSVKDNSKRYVVAGDLV GTKAKK SrtAPneu MESSHHHHHHAVLTSQWDAQKLPVIGGIAIPELEMNLPIFKGLDNVNLFYGAGTMK REQVMGEGNYSLASHHIFGVDNANKMLFSPLDNAKNGMKIYLTDKNKVYTYEIREV KRVTPDRVDEVDDRDGVNEITLVTCEDLAATERIIVKGDLKETKDYSQTSDEILTAFN QPYKQFY SrtALac MESSHHHHHHAAVKSVDFQSVLTNQFNSQPLPVIGGIAIPELNINLPIFKGVGNTS LLYGAGTMKADEVMGEGNYSLAGHNMTGFTSDLSILFTPLEKAKAGMTIYVTDKDN IYQYKIDKIDVVTPEHVEVINDSPGKKEITLVTCADAEATHRIIVHGTFVDKTSYDKAT DTMKNAFETKYNQIKNF SrtAFae MESSHHHHHHVESLSTEAVMKAQFENKNLPVIGAIAIPSVEINLPIFKGLSNVALL TGAGTMKEDQVMGKNNYALASHRTEDGVSLFSPLERTKKDELIYITDLSTVYTYKIT SVEKIEPTRVELIDDVPGQNMITLITCGDLQATTRIAVQGTLAATTPIKDANDDMLKA FQLEQKTLADWVA SrtAMono MESSHHHHHHGAANYDKDAVVGSIAVPSVDVNLLVFKGTNTANLLAGATTMRSDQ VMGKGNYPLAGHHMRDESMLFGPIMKVKKGDKIYLTDLENLYEYTVTETKTIDETE VSVIDDTKDARITLITCDKPTETTKRFVAVGELEKTEKLTKELENKYFPSK SrtASuis MESSHHHHHHSSGLVPRGSAILAAQWDAQRLPVIGGIAVPELGINLPIFKGVFNTSL MYGAGTMKENQEMGKGNYALASHHIFGVTGAADVLFSPLDRAKNGMKIYITDKTN VYTYVIDSVEIVSPESVYVIDDVEGRTEVTLVTCTDYYATQRIVVKGVLESTTPYNET AKDILDSFNKSYNQYDYG

67 SrtAOralis MESSHHHHHHSSGLVPRGSAVLASQWDAQKLPVIGGIAIPEVEINLPIFKGLDNVNL FYGAGTMKPDQKMGEGNYSLASHHIFTAENASQMLFSPLVNAKAGMKIYLTDKDK VYTYEITEVKRVTPDRVDEIEDRDGVKEITLVTCVDYNATERIIVKGIFKESKAYSETS EDILKAFNQPYRQRY

SrtAPlant (maltose binding protein fusion)

SrtAStrep (maltose binding protein fusion)

MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGD GPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAF KYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN YLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMSMDS SHHHHHHSSGLVPRGSVLQAQMAAQQLPVIGGIAIPELGINLPIFKGLGNTELIYGA GTMKEEQVMGGENNYSLASHHIFGITGSSQMLFSPLERAQNGMSIYLTDKEKIYEYI IKDVFTVAPERVDVIDDTAGLKEVTLVTCTDIEATERIIVKGELKTEYDFDKAPADVLK AFNHSYNQVST MBP-Kalata-B1 MHHHHHHKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQ VAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIA YPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNK GETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMP NIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNLEVLFNSSSNNNNNNNNNN LGIEGRISHMLEVLFQGPMGGGGSLPATGETCVGGTCNTPGCTCSWPVCTRNGL PVCGEG

Plasmids. The pET-28a+ expression vector for SrtAStaph was provided by Prof. Hidde

Ploegh (Whitehead Institute for Biomedical Research). The pET-15b expression vector for SrtAAnth was provided by Professor Robert Clubb (UCLA). Vectors for

SrtALac, SrtAPneu, SrtAFae, SrtAMono, and MBP-Kalata-B1 were obtained via

commercial gene synthesis (pJ414 expression vector) from DNA2.0 (Menlo Park, CA).

The catalytic domain of SrtAPlant was originally acquired through commercial

gene synthesis (pJ414 vector) from DNA 2.0. To improve solubility, the SrtAPlant

coding region was subcloned into the pMAL-C5x plasmid (New England Biolabs) to generate a maltose binding protein (MBP) fusion. Briefly, the SrtAPlant gene was

excised from the parent pJ414 vector by double digestion with EcoRI (Fisher OptizymeTM_{) and NcoI (Fisher Optizyme}TM_{) following the manufacturer’s instructions.}

The insert was then purified by agarose gel electrophoresis and isolated using a GeneJET Gel Extraction Kit (Thermo Scientific). pMAL-C5x was linearized and purified in a similar fashion. The SrtAPlant insert was then ligated using T4 DNA ligase

(Fisher OptizymeTM_{) following the manufacter’s instructions, and successful clones}

were confirmed by sequencing.

A pET-28a+ expression vector containing the catalytic domain of SrtAStrep was

provided by Prof. Hidde Ploegh (Whitehead Institute for Biomedical Research). Due to difficulties encountered during purification, the SrtAStrep coding region was

subcloned into the pMAL-C5x plasmid (New England Biolabs) to generate a maltose binding protein (MBP) fusion. First, the SrtAStrep gene was PCR amplified using the

Forward 5′-TATACCATGGACAGCAGCCATCATCATC-3′

Reverse 5′-GTCCGCTGAATTCTTAGGTAGATACTTGGTTATAAG-3′

A Phusion High-Fidelity PCR Kit (Thermo Scientific) was used for PCR following the manufacturer’s instructions. The PCR amplified product was then purified by agarose gel electrophoresis and isolated using a GeneJET Gel Extraction Kit (Thermo Scientific). The SrtAStrep insert was then cloned into pMAL-C5x via the NcoI

and EcoRI restriction sites, and successful clones were confirmed by sequencing. The catalytic domain of SrtASuis fused to a N-terminal His6-tag and flanked by

NcoI and EcoRI restriction sites was purchased as a GeneArt® StringTM _{(Invitrogen).}

This construct was digested using EcoRI and NcoI following the manufacturer’s instructions. The insert was then purified by agarose gel electrophoresis and isolated using a GeneJET Gel Extraction Kit (Thermo Scientific). The SrtASuis insert was then

cloned into the pJ414 backbone (DNA 2.0) via the NcoI and EcoRI restriction sites, and successful clones were confirmed by sequencing.

The catalytic domain of SrtAOralis fused to a N-terminal His6-tag and flanked by

NcoI and EcoRI restriction sites was purchased as a GeneArt® StringTM _{(Invitrogen).}

This construct was PCR amplified using the following primers:

Forward 5′-ATCGATCCATGGAAAGCA-3′

Reverse 5′-ATCGATGAATTCTTAATAACGCT-3′

A Phusion High-Fidelity PCR Kit (Thermo Scientific) was used for PCR following the manufacturer’s instructions. The PCR amplified product was then purified by agarose gel electrophoresis and isolated using a GeneJET Gel Extraction Kit (Thermo Scientific). The SrtAoralis insert was then cloned into pJ414 (DNA2.0) via the

NcoI and EcoRI restriction sites, and successful clones were confirmed by sequencing.

General Protocol for Protein Expression. Expression vectors for sortase homologs and MBP-cyclotide fusions were transformed (heat shock) into Escherichia coli BL21(DE3) cells and plated on agar plates supplemented with appropriate antibiotics (100 µg/ml ampicillin or 50 µg/ml kanamycin). After overnight incubation at 37 o_C,

single colonies were selected and used to inoculate 100 mL starter cultures containing sterile LB media and appropriate antibiotics. A portion of the starter culture (~25 mL) was used to inoculate 1 L of sterile LB containing antibiotics (100 µg/ml ampicillin or 50 µg/ml kanamycin), and the cells were grown at 37 o_{C to an}

optical density of ~0.8 at 600 nm. Protein expression was induced with IPTG (1 mM final concentration) for 3 hours at 37 o_{C. The cells were then harvested by}

centrifugation, followed by resuspension of the bacterial pellets in ~40 mL of buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 0.5 mM EDTA). Lysozyme was then added to a final concentration of 1 mg/ml, and the mixture was incubated for 2 h at 4 o_{C on}

a rocking platform. The suspension was then sonicated and centrifuged. The clarified lysate was then applied to a nickel nitrilotriacetic acid (Ni-NTA) column containing 5 mL of Ni-NTA agarose slurry (Thermo Scientific) that had been pre- equilibrated with ~10 mL of 20 mM Tris pH 8.0, 150 mM NaCl, and 20 mM imidazole. After binding proteins to the column, it was washed with 30 mL of 20 mM Tris pH 8.0, 150 mM NaCl, and 20 mM imidazole. Proteins were then eluted with three 5 mL portions of 20 mM Tris pH 8.0, 150 mM NaCl, and 300 mM imidazole. Purified

proteins were dialyzed against either 20 mM Tris pH 7.5, 150 mM NaCl (MBP- cycloide fusions) or 20 mM Tris pH 8.0, 150 mM NaCl (all sortase homologs) at 4 o_C

to remove imidazole. Glycerol was then added to the dialyzed solutions (final glycerol concentration 10% (v/v)), and aliquots were stored at -80 o_{C. All protein}

concentrations were estimated by absorbance at 280 nm using calculated extinction coefficients (Expasy ProtParam tool). The purity of each protein was evaluated by SDS-PAGE analysis.

5.2 Peptide Synthesis and Purification

Materials and Instrumentation. Unless otherwise noted, all chemicals were obtained from commercial sources and used without further purification. Fmoc Rink Amide MBHA resin and a colorimetric ninhydrin test kit were purchased from Anaspec. Fmoc-L-Ala-OH, Fmoc-L-Gly-OH, Fmoc-L-Ser(OtBu)-OH, Fmoc-L-Thr(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Ile-OH, Fmoc-L-Nle-OH, Fmoc-L-Pro- OH, Fmoc-L-Phe-OH, Fmoc-L-Tyr(OtBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L- Glu(OtBu)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Cys(Trt)- OH, Boc-2-aminobenzoic acid and O-(Benzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate (HBTU) were purchased from Chem- Impex International. Piperidine was obtained from Alfa Aesar. N,N′- diisopropylethylamine (DIPEA), peptide synthesis grade trifluoroacetic acid (TFA), dimethyl sulfoxide (DMSO), hydroxylamine hydrochloride, and methylene chloride (CH2Cl2) were obtained from Fisher Scientific. N-methyl-2-pyrrolidone (NMP) was

purchased from either Fisher Scientific or Advanced Chemtech. Fmoc-L-Lys(DNP)- OH was obtained from Anaspec of Chem-Impex International. Dithiothreitol (DTT) was purchased from Gold Biotechnology. Triisopropylsilane (TIPS) were obtained from Acros. Diethyl ether was purchased from J.T. Baker. ChromAR® grade acetonitrile (MeCN) was obtained from Macron Fine Chemicals. Water used in biological procedures or as a reaction solvent was purified using a Milli-Q Advantage A10 system (Millipore).

HPLC purifications and analyses were achieved using a Dionex Ultimate 3000 HPLC system. Analytical scale separations were performed with a Phenomenex Kinetex 2.6 μm, 100 Å C18 column (3.0 x 100 mm). Analytical scale analyses were achieved using the following method (Method A): MeCN (0.1% TFA) / H2O (0.1% TFA) mobile phase. Flow rate = 0.7 mL/min. Gradient = 10% MeCN (0.0-

0.5 min), 10% MeCN  90% MeCN (0.5-6.0 min), hold 90% MeCN (6.0-7.0 min), 90% MeCN  10% MeCN (7.0-7.1 min), re-equilibrate at 10% MeCN (7.1-10.0 min). Semi-preparative purifications were performed with a Phenomenex Luna 5 μm, 100 Å C18 column (10 x 250 mm). Semi-preparative purifications were achieved using the following method (Method B): MeCN (0.1% TFA) / H2O (0.1% TFA) mobile

phase. Flow rate = 0.5  4.0 mL/min (0.0-2.0 min), hold 4.0 mL/min (2.0-17.01 min), 4.0  0.5 mL/min (17.01-19.0 min). Gradient = 20% MeCN (0.0-2.0 min), 20% MeCN  90% MeCN (2.0-15.0 min), hold 90% MeCN (15.0-17.0 min), 90% MeCN

 10% MeCN (17.0-17.01 min), re-equilibrate at 10% MeCN (17.01-19.0 min). Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI- MS) analysis was carried out with an API 2000 Triple Quadrupole mass

spectrometer (Applied Biosystems) and an Agilent 1100 HPLC system equipped with a Phenomenex Aeris Widepore 3.6 μm 200 Å XB-C8 column (4.6 x 150 mm). All samples were analyzed using the following method: H2O (0.1% formic acid) /

organic (95% MeCN, 5% isopropanol, 0.1% formic acid) mobile phase. Flow rate = 1.25 mL/min. Gradient = 10% organic (0.0-1.0 min), 10% organic  90% organic (1.0-10.0 min). Deconvolution of protein charge ladders was achieved using a maximum entropy algorithm provided by Analyst 1.4.2 software.

UV-vis spectroscopy was performed on a NanodropTM_ND-1000

spectrophotometer (Thermo Scientific, USA).

General Peptide Synthesis Methods. All peptides were synthesized using manual Fmoc solid phase peptide synthesis (SPPS). Syntheses were performed in Extract CleanTM_{SPE 15 mL reservoirs fitted with appropriate frits and inlet/outlet caps. All}

manipulations (washing, coupling, deprotection, etc.) were conducted at room temperature and included gentle agitation on a bench-top rocking platform unless otherwise indicated.

Synthesis of Peptide Libraries using Isokinetic Couplings. Solid phase synthesis reservoirs were loaded with Fmoc-protected Rink amide MBHA resin (0.2 mmol scale). Resins were initially washed/swollen with ~10 mL of NMP (3x, 10 min per wash). Fmoc removal was achieved by treatment with 10 mL of 80:20 NMP/piperidine at room temperature (2x, 10 min per treatment). Resins were then washed with ~10 mL of NMP (3x, 5-10 min per wash). Peptide libraries were then

assembled using a combination of standard, single amino acid couplings and isokinetic couplings of amino mixtures. For single residue coupling the following procedure was employed: Fmoc-protected amino acid (0.60 mmol, 3.0 equivalents relative to resin loading), HBTU (0.60 mmol), and DIPEA (1.0 mmol) were dissolved in 3.0 mL of NMP. This solution was mixed thoroughly, and then added to the deprotected resin. Couplings were incubated for ~1 h at room temperature. Resins were then washed with ~10 mL of NMP (3x, 5-10 min per wash). The success of each coupling reaction was assessed using a colorimetric ninhydrin test. In the event that the coupling was incomplete, the amino acid coupling step was repeated. Fmoc deprotection was then carried out by washing with 10 mL of 80:20 NMP/piperidine (2x, 10 min per treatment), followed by washing with ~10 mL of NMP (3x, 5-10 min per wash). Repeated cycles of amino acid coupling and Fmoc deprotection were then performed to assemble the desired peptide sequences. Randomization of select positions using isokinetic amino acid coupling was achieved using a variation of the above coupling procedure. First, HBTU (2.0 mmol) and DIPEA (4.0 mmol) were dissolved in 10 mL of NMP along with the mixture of Fmoc-protected acids listed in the following Table 6.

Table 6. Composition of isokinetic mixture using in library synthesis.77

Amino Acid Building Block Mass (mg) mmol

Fmoc-Gly-OH 17 0.058 Fmoc-L-Ala-OH 21 0.068 Fmoc-L-Thr(OtBu)-OH 38 0.096 Fmoc-L-Ser(OtBu)-OH 21 0.056 Fmoc-L-Val-OH 77 0.226 Fmoc-L-Leu-OH 35 0.098 Fmoc-L-Ile-OH 123 0.348 Fmoc-L-Nle-OH 27 0.076 Fmoc-L-Tyr(OtBu)-OH 38 0.082 Fmoc-L-Trp(Boc)-OH 40 0.076 Fmoc-L-Asp(OtBu)-OH 29 0.070 Fmoc-L-Glu(OtBu)-OH 31 0.072 Fmoc-L-Asn(Trt)-OH 63 0.106 Fmoc-L-Gln(Trt)-OH 65 0.106 Fmoc-L-Pro-OH 29 0.086 Fmoc-L-Lys(Boc)-OH 71 0.124 Fmoc-L-Arg(Pbf)-OH 84 0.130 Fmoc-L-His(Trt)-OH 43 0.070

The coupling mixture was mixed until homogeneous, and then added to the deprotected resin. Isokinetic couplings were incubated for ~12 h at room temperature. Resin washing and Fmoc deprotection were then performed as described above. Additional Synthesis Notes: A commercially available lysine building block (Fmoc-L-Lys(DNP)-OH) was used to install the 2,4-dinitrophenyl

(DNP) chromophore. In addition, an aminobenzoic acid (Abz) fluorophore was installed at the N-terminus of all peptides through coupling of Boc-2-aminobenzoic acid. These building blocks were coupled following the standard single amino acid coupling procedure described above.

After assembly of the desired peptide sequences, the resins were washed with 10 mL of NMP (3x, 10 min per wash), followed by 10 mL of CH2Cl2 (3x, 10 min

per wash). Libraries were cleaved from the resin by treatment with 5 mL of 95:2.5:2.5 TFA/TIPS/H2O (2x, ~30 min per treatment). The combined cleavage

solutions were then concentrated on a rotary evaporator. The remaining residues were then precipitated from 40 mL of rapidly stirring, dry-ice chilled diethyl ether. The precipitated peptides were then collected by centrifugation, and the crude products were dried overnight under vacuum. This material was used without further purification. The presence of all predicted peptide sequences was confirmed using LC-ESI-MS. Peptide libraries were then dissolved in 1:1 DMSO/H2O, and total

peptide substrate concentrations were estimated using the absorbance of the DNP chromophore at 365 nm (extinction coefficient = 17,400 M-1_cm-1_{). Concentrations}

were then adjusted to yield final stock solutions containing 2 mM total peptide substrate.

Synthesis of Discrete Peptide Substrates. Solid phase synthesis reservoirs were loaded with Fmoc-protected Rink amide MBHA resin (0.1 mmol scale). Resins were initially washed/swollen with ~10 mL of NMP (3x, 10 min per wash). Fmoc removal was achieved by treatment with 10 mL of 80:20 NMP/piperidine at room temperature

(2x, 20 min per treatment). Resins were then washed with ~10 mL of NMP (3x, 5-10 min per wash). Suitably-protected amino acids were then coupled as follows: Fmoc- protected amino acid (0.30 mmol, 3.0 equivalents relative to resin loading), HBTU (0.30 mmol), and DIPEA (0.5 mmol) were dissolved in 2.5 mL of NMP. This solution was mixed thoroughly, and then added to the deprotected resin. Couplings were incubated for ~1 h at room temperature. Resins were then washed with ~10 mL of NMP (3x, 5-10 min per wash). The success of each coupling reaction was assessed using a colorimetric ninhydrin test. In the event that the coupling was incomplete, the amino acid coupling step was repeated. Fmoc deprotection was then carried out by washing with 10 mL of 80:20 NMP/piperidine (2x, 10 min per treatment), followed by washing with ~10 mL of NMP (3x, 5-10 min per wash). Repeated cycles of amino acid coupling and Fmoc deprotection were then performed to assemble the desired peptide sequence. Additional Synthesis Notes: A commercially available lysine building block (Fmoc-L-Lys(DNP)-OH) was used to install the 2,4-dinitrophenyl (DNP) chromophore. In addition, an aminobenzoic acid (Abz) fluorophore was installed at the N-terminus of all peptides through coupling of Boc-2-aminobenzoic acid. These building blocks were coupled following the standard single amino acid coupling procedure described above.

After assembly of the desired peptides, the resins were washed with ~10 mL of NMP (3x, 10 min per wash), followed by ~10 mL of CH2Cl2 (3x, 10 min per wash).

Peptides lacking cysteine or methionine were cleaved from the resin using a cleavage cocktail consisting of 95:2.5:2.5 TFA/TIPS/H2O. For peptides containing

as a solid to give a final concentration of 5% w/v) was employed. Peptide cleavage was achieved by treating resins with 5 mL of the appropriate cleavage cocktail (2x, ~30 min per treatment). The combined cleavage solutions were then concentrated on a rotary evaporator. Crude peptides were then precipitated from 40 mL of rapidly stirring, dry-ice chilled diethyl ether. The precipitated peptides were then collected by centrifugation, dried overnight under vacuum. All peptides were purified by RP- HPLC (Method B). Pure fractions were pooled and lyophilized. The identity and purity of all peptides was confirmed by RP-HPLC and LC-ESI-MS. Peptides were then dissolved in 1:1 DMSO/H2O, and peptide concentrations were estimated using

the absorbance of the DNP chromophore at 365 nm (extinction coefficient = 17,400 M-1_cm-1_{). Concentrations were then adjusted to yield 2 mM stock solutions.}

5.3 Peptide Library Screening and Peptide Model Reactions

General Protocol for Sortase-Catalyzed Transacylations using Isokinetic Libraries and Discrete Peptide Substrates. Reactions were generally conducted on 50 μL scale. The stock solutions shown below (Table 7) were combined in appropriate ratios in microcentrifuge tubes, and then diluted with water to give the indicated reactant concentrations. All reactions were incubated at room temperature unless otherwise indicated. All reactions contained 10% (v/v) 10x SrtA reaction buffer (500 mM Tris pH 7.5, 1500 mM NaCl, 100 mM CaCl2). Additional notes: the sortase stock

solutions contained 10% (v/v) glycerol, and therefore all reactions contained small amounts of glycerol ≤ 6% (v/v). Additionally, the stock solutions for all peptides were prepared in 1:1 DMSO / H2O, and therefore all reactions employing these materials

contained DMSO at a final concentration of 5% (v/v). Reactions were analyzed by RP-HPLC and LC-ESI-MS. For RP-HPLC, unless otherwise indicated reactions were directly injected into the HPLC system and separated using Method A (see above). Estimates of reaction progress were obtained by comparing peak areas derived from the 365 nm chromatogram. For LC-ESI-MS, reactions were directly injected using the method described above.

Table 7. Stock solutions used in sortase-mediated peptide ligation reactions:

Reagent Concentration Solvent/Buffer

Abz-LPATXG-K(DNP) 2mM 1:1 DMSO/Water

Abz-LPAXGG-K(DNP) 2mM 1:1 DMSO/Water

Abz-LXATGG-K(DNP) 2mM 1:1 DMSO/Water

Abz-XPATGG-K(DNP) 2mM 1:1 DMSO/Water

All Discrete Peptides 2 mM 1:1 DMSO/Water

H2NOHHCl 100mM Water

GG 10mM Water

DTT 1 M Water

β-ME 100 mM Water

SrtAstaph 444 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAsuis 115 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAoralis 220 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtApneu 89 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAmono 248 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAfaec 354 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAlac 139 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAstrep 44 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAanth 451 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

SrtAplant 281 µM 90% 50mM Tris pH 8.0, 150 mM NaCl, 10%

Glycerol (v/v)

10x SrtA reaction buffer - 500 mM Tris pH 7.5, 1500 mM NaCl, 100 mM

80 5.4 Kalata B1 Synthetic Studies

One-Pot Kalata B1 Cyclization using Sortase Homologs. MBP-Kalata-B1 (14.6 µL of a 171 µM stock solution in 20 mM Tris pH 7.5, 150 mM NaCl, 10% (v/v) glycerol) was combined with DTT (5.0 µL of a 1 M stock solution in H2O), H2NOHHCl (5.0 µL

of a 100 mM stock solution in H2O), and 10x sortase reaction buffer (5.0 μL of 500

mM Tris pH 7.5, 1500 mM NaCl, 100 mM CaCl2). Appropriate amounts of each

sortase homolog were then added to bring the final enzyme concentration to 25 µM. H2O was then added to bring the final volume to 50 μL, and reactions were

incubated for 24 hours at 37 o_{C. Reactions were then analyzed by LC-ESI-MS and}

In document Profiling Sortase Substrate Specificity using Peptide Libraries (Page 76-91)