fluorescence anisotropy assay for proteolysis of specifically labeled fusion proteins

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Analytical Biochemistry 336 (2005) 75–86

ANALYTICAL

BIOCHEMISTRY

Fluorescence anisotropy assay for proteolysis of specificallylabeled fusion proteins

Paul G. Blommel, Brian G. Fox*

The University of Wisconsin Center for Eukaryotic Structural Genomics, Biophysics Graduate Degree Program

and Biochemistry Department, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA

Received 9 July 2004

Abstract

A cloning method and plasmid vectors that permit fluorescence-anisotropy-based measurement of proteolysis are reported. Therecombinant protein substrates produced by this method contain a tetracysteine motif that can be site-specifically labeled with bis-arsenical fluorophore [Science 281 (1998) 269]. Six protein substrates with an N-terminal fusion of the tetracysteine motif and dif-ferent protease recognition sites were created and tested for reaction with commercial proteases commonly used to process recom-binant fusion proteins. In each case, proteolysis of a single susceptible peptide bond could be monitored in real time and withsufficient data quality to allow numerical analysis of proteolysis reaction kinetics. Measurement of proteolysis extent using fluores-cence anisotropy is shown to be comparable to densitometry measurements made on denaturing polyacrylamide gels but with theadded advantages implicit in a time-resolved measurement, quantification by a spectroscopic measurement, and facile extensibilityto high-throughput formats. The assay was also demonstrated as a general tool for monitoring proteolysis of multidomain fusionproteins containing an internal protease site such as are being created in structural genomics studies worldwide.� 2004 Elsevier Inc. All rights reserved.

Keywords: Protease assay; Protease substrate; Fluorescence anisotropy; FLAsH; Fusion tags; TEV protease; Fusion protein; Structural genomics;Tetracysteine; TetraCys; bis-Arsenical fluorophore; High-throughput assay

Numerous methods for the assay of proteases havebeen developed [1]. These may rely on small moleculesor peptide analogs of protease substrates monitoredcontinuously through spectrophotometric changes thatoccur after scissile bond cleavage [2,3]. Assays of thistype have proven useful for exploring protease reactionmechanisms and for determining primary sequence spec-ificity but fail to address issues of recognition sequenceaccessibility or protease–protein interactions that areimportant for folded protein substrates.

There are also a number of options available to assaythe proteolysis of folded protein substrates. Unfortu-nately, these are usually limited to measurement at dis-

0003-2697/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2004.09.023

* Corresponding author. Fax: +1 608 262 3453.E-mail address: [email protected] (B.G. Fox).

crete time points using high-performance liquidchromatography [4], capillary electrophoresis [5], poly-acrylamide gel electrophoresis [6], or other approachesrequiring time-consuming analysis of quenched reac-tions. Protease assays based on fluorescence anisotropy(also known as fluorescence polarization) measurements[7,8] represent an exception to this limitation. Thesefluorescence-based assays can be monitored continu-ously and are suitable for high-throughput applications[7]. Indeed, fluorescence anisotropy has been used tomeasure the activity of nonspecific proteases when ki-netic data for a single well-defined cleavage event arenot required. However, since conventional fluorophorelabeling reagents will nonspecifically label free func-tional groups such as thiols or amines, fluorescent labelsare likely to be introduced at multiple locations within aprotein. This results in a heterogeneous substrate for

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76 Fluorescence-anisotropy-based measurement of proteolysis / P.G. Blommel, B.G. Fox / Anal. Biochem. 336 (2005) 75–86

which the detected change in fluorescence anisotropy isdue to the sum of changes in rotational correlation timeof multiple fluorophores at different positions within theprotein. Moreover, as proteolysis proceeds, multiplefragments of protein are released, each of which be-comes a potential new substrate for proteolysis, compli-cating kinetic analysis.

An emerging biotechnological application of prote-ases is the proteolysis of recombinant fusion proteins.This approach is widely applied as an enabling technol-ogy for structural biology [9,10]. In many cases, the pro-tein purification process is based on production of adesired target protein as a fusion with an affinity tagsuch as His6 [11] and a solubility domain such as malt-ose binding protein, thioredoxin, glutathione S-transfer-ase, or others [12,13]. The affinity tag and solubilitydomain are removed after purification by proteolysisat a protease recognition site engineered between the sol-ubility domain and the target protein. Methods thatprovide for detection of fusion proteins and real-timemonitoring of proteolysis in a high-throughput formatwould then be of potential utility. During the courseof our work, we considered that introduction of the tet-raCys motif [14] (CCPGCC) could potentially be ap-plied to these problems. Through incorporation of thismotif at an appropriate location within a recombinantfusion protein, site-specific labeling of the protein witha bis-arsenical fluorophore could be achieved [14]. Sim-ple measurement of fluorescence intensity of the labeledprotein would then provide a method for protein detec-tion and quantification. Furthermore, by measuringchanges in fluorescence anisotropy, we proposed tomonitor the release of a small peptide containing the flu-orescently labeled tetraCys motif from proteolysis of thecomplete fusion protein. Along with rate data, the addi-tive nature of anisotropy measurements would permit areal-time evaluation of the percentage progress of theproteolysis reaction.

Herein, we describe the use of the tetraCys motif toenable the measurement of specific proteolysis of nativeprotein substrates. The method was investigated usingN-terminal-modified forms of Escherichia coli maltosebinding protein (MBP)1 as substrates for four proteasescommonly used for removal of fusion tags from targetproteins: thrombin, Factor Xa, enterokinase, and TEVprotease. Trypsin was also tested as an example nonspe-cific protease. In all cases, the initial proteolysis ratesdetermined by fluorescence anisotropy were linear withrespect to protease concentration and correlated withthe extent of proteolysis indicated by SDS–PAGE anal-ysis. The assay was also demonstrated for the proteo-lytic processing of two unknown Arabidopsis thaliana

1 Abbreviation used: MBP, maltose binding protein.

proteins expressed from plasmids encoding an internaltetraCys motif as part of a larger multidomain recombi-nant fusion protein.

Materials and methods

Materials

Thrombin, enterokinase, Factor Xa, and the E. coli

expression strain Rosetta were from Novagen (Madison,WI). Trypsin was from Calbiochem (San Diego, CA).Vectors pDEST17 and pDONR221, all Gateway re-agents, E. coli DB3.1a and Top 10 competent cells,and the FLAsH labeling reagent were obtained fromInvitrogen (Carlsbad, CA). Plasmid vector pQE80 camefrom Qiagen (Valencia, CA). DNA for amplification ofE. coli MBP was derived from vector pMAL-C2 (NewEngland Biolabs, Beverly, MA). The ExSite site-directedmutagenesis materials and Yield Ace DNA polymerasewere from Stratagene (La Jolla, CA). All PCR primerswere obtained from IDT (Coralville, IA). Criterion12.5% Tris–HCl precast gels were used for SDS–PAGEanalysis (Bio-Rad, Hercules, CA) and stained using Bril-liant blue R (Sigma, St. Louis, MO). Assays were con-ducted in 384-well microplates, either Cliniplate brand(Thermo-Labsystems, Waltham, MA) or Greiner(Frickenhausen, Germany). Protein was concentratedusing Amicon Ultra concentrators (Millipore, Billerica,MA). TEV protease mutant S219V was refolded frompurified inclusion bodies [15,16].

Construction of pVP14

The pVP14 vector was created from pDEST17 usingthe ExSite mutagenesis procedure. The forward primer(FLUF) was 5 0 phosphorylated and consisted of the se-quence TGT TGC CCA GGA TGC TGT ACA AGTTTG TAC AAA AAA GCT GAA CGA GAA. The re-verse primer (pD17FR) was not 5 0 phosphorylated andconsisted of the sequence TGA TTC GAG GTG ATGGTG ATG GTG ATG GTA GTA CGA CAT AT.PCR was performed using Yield Ace polymerase in aDYAD DNA Engine thermocycler (MJ Research, Wal-tham, MA). The reaction cycle consisted of 5 min at94 �C, 1 min at 55 �C, 6 min and 45 s at 72 �C, followedby 14 cycles of 1 min at 94 �C, 1 min at 65 �C, 6 min and45 s at 72 �C, followed by 5 min at 72 �C and then 4 �Cuntil needed. The �6.5-kb amplified DNA fragment waspurified by agarose gel electrophoresis and ligated withT4 DNA ligase using a temperature program consistingof 30 s at 4 �C and 30 s at 30 �C repeated continuouslyfor �18 h. Competent E. coli DB3.1a cells were trans-formed with the ligation reaction mixture. Plasmidsrecovered from transformed colonies were sequencedto verify successful mutagenesis.

Table 1Forward primers used for incorporation of protease-susceptiblesequences

21-F 5 0 Internal primer AAC CTG TAC TTC CAG—MBP

5 0 External primer GW—GAA AAC CTG TAC TTC CAG

22-F 5 0 Internal primer TTC CTC GGC ATG GTC—MBP

5 0 External primer GW—CGC TCC TTC CTC GGC ATG GTC

23-F 5 0 Internal primer GAT GAC GAT GAC AAG—MBP

5 0 External primer GW—GAA GAT GAC GAT GAC AAG

24-F 5 0 Internal primer ATC GAA GGA CGC—MBP

5 0 External primer GW—ATC GAA GGA CGC

25-F 5 0 Internal primer GTA CCA CGT GGC AGT—MBP

5 0 External primer GW—CTA GTA CCA CGT GGC AGT

26-F 5 0 Internal primer AAC CTG TAC TTC CAG TCC—MBP

5 0 External primer GW—GAA AAC CTG TAC TTC CAG

MBP = AAA ATC GAA GAA GGT AAA CTG GTA ATC;GW = GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCC.

Fluorescence-anisotropy-based measurement of proteolysis / P.G. Blommel, B.G. Fox / Anal. Biochem. 336 (2005) 75–86 77

Construction of pVP15

The plasmid pVP13, a pQE-80-derived vector usedfor expression of A. thaliana genes in E. coli at Univer-sity of Wisconsin Center for Eukaryotic StructuralGenomics, was used as the parent for production ofpVP15. Proteins expressed from pVP13 contain an Stag for detection [17], a His6 tag for purification, andMBP to enhance target protein folding. To producepVP15, the linker peptide between the MBP and theattB1 sequence of pVP13 was modified to include aTEV protease cleavage site, a His6 tag, and the tetraCysmotif. The second His6 tag was added to allow bindingof the tetraCys motif during subtractive IMAC purifica-tion after proteolysis [18]. To modify pVP13, the FLUFforward primer described above was used. The reverseprimer (p13HTF) consisted of the sequence ATGGTG GTG GGA CTG GAA GTA CAG GTT TTCGTT GTT GTT CGA GCT CGA ATT AGT CTGCGC GTC TTT CAG GGC TTC. Thermocycling wasconducted as described for the construction of pVP14with the exceptions that the annealing temperature was69 �C, 12 cycles were used instead of 14, and the ampli-fied band was not gel-purified before ligation.

Cloning methods

All cloning was completed using the Gateway Systemfor recombination cloning. A nested PCR was used toincorporate different protease cleavage sites betweenthe attB1 sites and E. colimaltose binding protein. Table1 shows sequences of the primers used for PCR in the 5 0

to 3 0 direction, while Fig. 1 shows a schematic represen-tation of the two-step PCR used to create DNA insertsfor recombination into the attR recombination sites ofeither pVP14 or pVP15. All forward primers for thefirst-step PCR consisted of nucleotides required for theprotease cleavage site followed by 27 nucleotides identi-cal to the MBP gene sequence. Reverse primers wereidentical for all substrates and consisted of the sequenceshown in Fig. 1. The MBP template material was de-rived from the vector pMal-C2. The first PCR incorpo-

Fig. 1. Schematic and example of nucleotide sequence overlaps used to generaTEV protease site between the tetraCys motif and MBP. Other protease sites

rated 5 0 and 3 0 internal primers (see Table 1 and Fig. 1)and consisted of 5 min at 95 �C, 21 cycles of 30 s at94 �C, 30 s at 55 �C, 1 min and 30 s at 72 �C, followedby 7 min at 72 �C and then 4 �C until needed. For thesecond PCR, 10 lL of the first PCR was transferred toa second 50-lL PCR containing fresh nucleotides,DNA polymerase, reaction buffer and 5 0 and 3 0 externalprimers (see Table 1 and Fig. 1). The conditions for thesecond PCR were identical to the first. The PCR prod-uct was subjected to a BP recombination reaction withpDONR221. The recombination product was trans-formed into E. coli Top 10.

The recombination region of plasmids isolated frompositive transformants was sequenced before beingtransferred by the LR reaction to the destination vectorpVP14. This recombination created the His6-Cys4-X-MBP constructs used for protein expression, where Xrepresents an engineered protease recognition site. TheN-terminal amino acid sequences of the expressed fusionproteins are shown in Table 2.

Arabidopsis open reading frames At3g16990 andAt3g03410 were cloned using a protocol identical to thatdescribed above with the exception that the sequencesspecific for these genes were substituted for the MBP se-quence. The PCR products of the two-step amplificationwere transferred to pDONR221 and sequenced. Verifiedsequence clones were transferred into either pVP14 or

te coding sequences for protease substrates. The example shown places awere created by use of the oligonucleotide primers indicated in Table 1.

Table 2Amino acid sequences of various His6-Cys4-X-MBP protease substrates

Substrate ID Intended protease susceptibility Sequencea

21-F Noneb MSYYHHHHHHLESCCPGCCTSLYKKAGSENLYFKS-MBPc

22-F Multipled MSYYHHHHHHLESCCPGCCTSLYKKAGSRSFLGMV-MBP23-F Enterokinase MSYYHHHHHHLESCCPGCCTSLYKKAGSEDDDDK›-MBP24-F Factor Xa MSYYHHHHHHLESCCPGCCTSLYKKAGSIEGR›-MBP25-F Thrombin MSYYHHHHHHLESCCPGCCTSLYKKAGSLVPR›GS-MBP26-F TEV protease MSYYHHHHHHLESCCPGCCTSLYKKAGSENLYFQ›S-MBP

a The tetraCys motif used for binding the FLAsH reagent is highlighted in gray. The underlined residues make up the protease recognition site.The susceptible peptide bond found in each protease site is indicated with an arrow.

b The TEV site was substituted with Lys in the P10 position. Previous studies have shown that this alteration to the TEV site results in a loss ofproteolysis [22].

c The first 10 residues in the N-terminal sequence of MBP are KIEEGKLVIW.d The substrate 22-F contains cleavage sites for multiple proteases, including enterokinase, Factor Xa, and thrombin. A TEV protease recognition

site was not present.


pVP15 using the LR reaction to create expressionplasmids.

Expression and purification of protease substrates

All protease substrates were expressed in E. coli Ro-setta pLacI Rare to provide compensation for rare co-dons [19]. The expression was done in 2-L PET bottles[20]. The protein purification followed typical IMACpurification procedures [18] including elution in an imid-azole gradient from 0 to 350 mM. After SDS–PAGEanalysis, appropriate fractions were pooled and desaltedinto 20 mM phosphate, pH 7.5, containing 100 mMNaCl and 0.3 mM TCEP. Proteins were concentratedto a final concentration of 300–600 lM using centrifugalconcentrators. Glycerol (50% v/v) was added to all pro-tease substrates except 26-F and frozen at �20 �C. TheArabidopsis targets At3g03410 expressed from pVP14and At3g16990 expressed from pVP15 were flash-frozenas drops in liquid nitrogen and stored at �80 �C.

FLAsH labeling reactions

A 20% molar excess of the FLAsH reagent was incu-bated overnight (�12 h) with protein (10–100 lg) atroom temperature in 20 mM phosphate, pH 7.5, con-taining 100 mM NaCl, 0.3 mM TCEP, and 1 mMb-mercaptoethanol. After the labeling reaction, unincor-porated fluorophore was removed by two successive cy-cles of 10-fold dilution and concentration usingcentrifugal concentrators.

Protease assays

Protease assays were conducted using a combinationof 20 lM unlabeled and 40 nM labeled protease sub-strates. TEV protease reactions were conducted in20 mM Tris–HCl, pH 8.0, containing 100 mM NaCl,0.3 mM TCEP, 1 mM b-mercaptoethanol, and 5 mM

EDTA. Reactions with thrombin, enterokinase, and Fac-tor Xa were performed in the buffer supplied by the man-ufacturer. Trypsin reactions were carried out in the buffersupplied for thrombin. Assays were carried out in blackCliniplate 384-well plates in a 40-lL reaction volume orin black Greiner 384-well plates using an 80-lL reactionvolume. Equal volumes of substrate and protease dilutedin reaction buffer weremixed in the assay plates to initiatereactions. Kineticmeasurements were begun immediatelyafter mixing. To avoid a long time delay between the mix-ing and the first timemeasurement, a maximum of 135 as-says were conducted in parallel. This decreased theamount of time between mixing and measurement to lessthan 3 min and allowed the fluorescence anisotropy mea-sured at the first time point to closely approximate theanisotropy of the intact fusion protein. The reaction tem-perature was maintained between 23 and 28 �C.

Fluorescence anisotropy measurements

Fluorescence anisotropy was calculated asr = (f^ � fi)/(f^ + 2 fi) where f^ is fluorescence intensitymeasured perpendicular to the excitation and fi is theintensity measured parallel to the excitation light.Anisotropy values were presented as mr = 1000 r. Themeasurements were made using a Tecan 384 Ultra platespectrofluorimeter. Excitation was at 485 nm (25 nmbandpass) and emission was measured at 525 nm(20 nm bandpass). Anisotropy measurements were madeat 2- to 5-min intervals for 5 h. The observed anisotropyis the sum of the anisotropy of the individual fluorescentcomponents, or robs =

Pfi ri, where robs is the observed

anisotropy, fi is the fractional fluorescence intensity ofcomponent i, and ri is the anisotropy of component i

[21]. For a two-state system with the fluorophore at-tached to either the intact fusion protein or the smallpeptide produced by proteolysis, the fractional extentof reaction is given by n = (rf � robs)/(rf � rc), where rfis the fluorescence anisotropy of the intact fusion pro-


tein and rc is the fluorescence anisotropy of the smallpeptide. This treatment assumes that there is no differ-ence in the fluorescence intensity of the fluorophore at-tached to either the fusion protein or the smallpeptide, and this is justified by the observation that therewas no significant change in total fluorescence intensityas the reactions proceeded. The reaction progress curveswere fit with either exponential or linear models depend-ing on the extent of reaction at the end of the measure-ment period. For less than 20% reaction at the end of themeasurement period, a linear model was sufficient to de-scribe the initial reaction rate. Above 20% reaction, anexponential decay model was used. XLfit3 (ID BusinessSolutions, Guildford, UK) was used for curve fittingand statistical analysis. Errors are reported as two stan-dard deviations.

Scanning densitometry of SDS–PAGE gels

The intensity of protein bands in SDS–PAGE gels wasdetermined using Image J (version 1.30; public domainsoftware available from http://rsb.info.nih.gov/ij/).

Results

Expression vectors pVP14 and pVP15

Fig. 2 shows schematic representations of the fusionproteins described here. The expression vector pVP14

Fig. 2. Schematic representations of fusion proteins created fromexpression vectors pVP14 and pVP15 and the peptides released afterproteolysis. (A) Expression vector pVP14 creates an N-terminal fusionconsisting of His6, the tetraCys motif required for labeling with bis-arsenical fluorophores, the amino acids encoded by the attB1 recom-bination site, the desired protease recognition site, and the target gene.Proteolysis releases the small N-terminal peptide (�4 kDa) thatcontains the fluorescently labeled tetraCys motif. The resulting changeof fluorescence anisotropy can be monitored to determine the extent ofproteolysis. (B) Expression vector pVP15 creates a fusion proteinconsisting of S tag, His6, MBP, the TEV protease recognition site, thetetraCys motif, the amino acids encoded by the attB1 recombinationsite, a second protease recognition site, and the target gene. Proteolysisat both protease sites will release the small internal peptide (�3 kDa)that contains the fluorescently labeled tetraCys motif.

(Fig. 2A) encodes an N-terminal His6 affinity tag, thetetraCys (CCPGCC) sequence for site-specific labelingwith FLAsH reagent, and the attB1 site-specific recom-bination site. Upon proper preparation of a DNA insert(described below), a fusion protein consisting of the vec-tor-encoded protein sequences, a protease recognitionsequence, and a desired target protein can be expressed.For the methods development work reported here, E.coli MBP was used as a convenient highly soluble targetprotein. Treatment of the fusion protein obtained frompVP14 with the appropriate protease would release a�4-kDa fluorescent peptide from the N-terminal of thedesired target protein.

Fig. 2B shows a schematic representation of a fusionprotein produced from pVP15. This expression vector en-codes an N-terminal S tag and His6 affinity tag, MBP, aTEV protease site, a second His6 affinity tag, the tetraCysmotif, and the attB1 sequence. Upon proper insertion of acloned gene into the recombination sites, a fusion proteincontaining an internal tetraCys motif bound by two pro-tease sites is created (Fig. 2B). Treatment of this fusionprotein with the appropriate combination of proteaseswould release a �3-kDa fluorescent peptide from the lin-ker peptide between the S-tag-His6-MBP solubility do-main and the desired target protein.

Substrate design

The two-step PCR amplification using the primersshown in Table 1 creates a DNA fragment that can beefficiently recombined into either pVP14 or pVP15. Atthe 5 0 end of the fragment, the first PCR primer dupli-cates 27 nucleotides of the desired target gene and pro-vides part of the desired protease recognition site. Thesecond PCR primer completes the desired protease rec-ognition site and provides the attB1 recombination se-quence. At the 3 0 end, the first PCR primer duplicates20 nucleotides of the desired target gene including thestop codon, while the second PCR primer provides theattB2 sequence. In vitro recombination of the amplifiedfragment with either the pVP14 or the pVP15 vectorthen creates an expression vector encoding a fusion pro-tein consisting of one or more affinity tags, a solubilitydomain (with the use of pVP15), and protease sites re-quired to remove the tags from the target protein afterexpression, purification, and treatment with the appro-priate protease. This primer-based design was used tocreate six fusion protein constructs that differed onlyin the identity of the protease recognition site (Table2). The substrates with the different protease-susceptiblesites are designated X-MBP.

The vector pVP14 was used to create fusion proteinssuitable for testing the applicability of fluorescenceanisotropy to specific protease reactions. By variationof the nucleotide sequence in the forward primers, rec-ognition sites for TEV protease, enterokinase, Factor

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Xa, and thrombin were placed between the N-terminaltetraCys motif and the MBP. These vectors allowed pro-duction of substrate-level amounts of the purified fusionprotein (yields of >100 mg/liter) from standard bacterialculture, expression, and IMAC affinity purification.These fusion proteins are schematically represented asHis6-Cys4-X-MBP.

Fig. 3. Time-dependent changes in fluorescence anisotropy measuredwith two different FLAsH-labeled substrates incubated with theappropriate proteases. (A) Substrate 25-F incubated with threedifferent thrombin concentrations, � 0.26 lM, m 0.07 lM, and ·0.004 lM, and d control with no protease. Measurements were takenin triplicate with error bars representing one standard deviation aboveand below the mean. (B) Substrate 26-F incubated with threeconcentrations of TEV protease: � 1.4 lM, m 0.35 lM, · 0.09 lM,and d control with no protease. Measurements were taken inquadruplicate with error bars representing one standard deviationabove and below the mean. Measurements were taken at 3-minintervals, while data points are shown at 9-min intervals for clarity.

Use of fluorescent-labeled substrates to determine

protease activity

Fig. 3 shows primary fluorescence anisotropy dataobtained during the reactions of thrombin (Fig. 3A)and TEV protease (Fig. 3B) with their respective suscep-tible substrates 25-F and 26-F. In the absence of prote-ase, both FLAsH-labeled His6-Cys4-X-MBP substrateswere stable and gave a relatively constant anisotropyconsistent with the size of the intact fusion protein.Incubation of the substrate with the appropriate prote-ase gave a time-dependent decrease in fluorescenceanisotropy consistent with release of the small labeledpeptide from the larger fusion protein.

Fig. 4 shows a rearrangement of the primary anisot-ropy data into a progress curve for the reaction ofTEV protease with substrate 26-F. The progress curvewas well fit by a first order exponential except at thelow reaction rate observed for low protease concentra-tions. In this case, where the extent of reaction was lessthan 20% at the end of the measurement period, a linearapproximation was sufficient to fit the progress curve.

Progress curves obtained for the other proteases weresimilar and could be fit by a first order exponential todetermine first order decay rates. These initial reactionrates are plotted versus protease concentration in Fig.5. For trypsin, thrombin, and TEV protease, the concen-trations of the protein preparations were known and aspecific reaction rate was calculated from the slope ofthe plot of initial reaction rates versus protease concen-tration (Fig. 5A). The specific reaction rates are listed inthe caption to Fig. 5. For thrombin, Factor Xa, andenterokinase, activities (U/lL) were reported by the sup-

Fig. 4. Fractional conversion data for substrate 26-F incubated withdifferent concentrations of TEV protease: � 1.4 lM, m 0.35 lM, and ·0.09 lM.Measurements were taken at 3-min intervals, while data pointsare shown at 9-min intervals. The solid lines represent first orderexponential fits to the entire experimental data set. Four replicates ofexperimental data were used for fractional conversion calculations.Error bars represent one standard deviation above and below the mean.

Fig. 5. Initial proteolysis rate versus protease concentration for fivecombinations of FLAsH-labeled substrate and protease. (A) Compar-ison of the proteolysis rates for trypsin, thrombin, and TEV protease.The series represented are j trypsin with substrate 22-F, · thrombinwith substrate 25-F, and � TEV protease with substrate 26-F. Specificreaction rates were calculated based on the slope of linear least squarescurve fits shown as solid lines.

Fig. 6. Comparison of the extent of proteolysis determined by eitherfluorescence anisotropy or by SDS–PAGE with Coomassie stainingand densitometry measurements. The solid line is a linear least squaresfit; r2 = 0.99. Specific reaction rates in units of moles substrateproteolyzed per mole of protease per hour were as follows: trypsin,720 ± 60; thrombin 270 ± 6; and TEV protease, 17.3 ± 0.7.


plier as the amount of a standard fusion protein prote-olyzed per unit time. The plot of initial reaction rate ver-sus standard units added to the anisotropy assay isshown in Fig. 5B for these proteases. For all proteasestested, the initial reaction rates were found to be linearwith respect to protease concentration except with thehighest levels of trypsin and TEV protease (measure-ments deviating from linearity are not shown in Fig. 5).

Comparison of anisotropy and SDS–PAGE

By evaluation of the primary anisotropy results asshown in Fig. 3, the fractional extent of proteolysis, n,

can be determined (see Materials and methods). Like-wise, SDS–PAGE coupled with Coomassie staining hasbeen widely applied to determine the extent of proteolysisof a fusion protein [22], as the fractional extent of reactioncan be approximated from the relative intensities of theintact fusion protein and the released target protein.Fig. 6 shows the equivalence of fluorescence anisotropyand SDS–PAGE as analytical methods to determine thefractional extent of proteolysis. It is noted that the anisot-ropy measurement arises from a numerical analysis of atime-resolved measurement, while the SDS–PAGEmeth-od is time-discontinuous and requires sample prepara-tion, electrophoresis, gel staining, and gel imaging.

Cross-reactivity of proteases

The complete set of His6-Cys4-X-MBP substrates wasinvestigated for cross-reactivity under the reaction con-ditions used. Fig. 7 shows representative raw anisotropydata for the general protease substrate 22-F and thethrombin substrate 25-F. Relative rates were determinedfor protease/substrate combinations by nonlinear leastsquares fitting of the primary data and normalizationwith the rate determined for the intended substrate ofeach protease. The relative rates are shown in Table 3.TEV protease was found to react with only 26-F, thesubstrate containing the consensus TEV protease cleav-age site. As expected, trypsin was found to proteolyze allof the substrates tested due to the introduction of Lysresidues by the attB recombination sequence used forcloning. Moreover, changes in anisotropy indicated that

Fig. 7. Incubation of FLAsH-labeled protease substrates with alter-native proteases to evaluate specificity. Example fluorescence anisot-ropy data are shown for reaction of substrate 22-F (A) and 25-F (B).Proteases tested include j 0.1 lM trypsin, · 0.13 lM thrombin, d0.11 U/ll enterokinase, � 2 lMTEV protease, andm 0.06 U/ll FactorXa, – negative control.

Table 3Relative rates of reaction for His6-Cys4-X-MBP substrates with different pro

Protease Substrateb

21F 22F 23

Trypsin 0.3 1 0Enterokinase 0.5 1.3 1Factor Xa 0.04 0.5 0Thrombin 0.0 0.4 -0TEV 0.01 0.0 -0

a The initial rates of reaction were determined by fluorescence anisotropyrates were calculated by normalization to the rate determined for the reactiotrypsin initial rates were normalized with respect to the initial rate determin

b N-terminal sequences of protease substrates given in Table 2.c Not determined.


each of the other three proteases gave variable amountsof substrate-dependent nonspecific proteolysis.

SDS–PAGEanalysiswithCoomassie stainingwas per-formed to further evaluate the predicted low rates of sub-strate-dependent nonspecific proteolysis. Fig. 8 showsthat Factor Xa reacted with the cognate substrate 24-F(lane 2) and the general protease substrate 22-F (lane 4),as little or no intact fusion protein was detected after anovernight incubation. Furthermore, the low rates of non-specific proteolysis suggested by the fluorescence anisot-ropy assay for reaction of Factor Xa with substrates21-F, 23-F, and 25-F (Table 3) were confirmed by SDS–PAGE through the formation of a small amount of asmall-molecular-weight band (seeFig. 8, lanes 1, 3, and 6).

For TEV protease, the TEV substrate with Lys in theP1 0 position, 21-F (Fig. 8, lane 7) underwent a minoramount of proteolysis, shown by both the anisotropyand the SDS–PAGE assays. Other studies have shownthat the substitution of Lys into the P1 0 position of anotherwise intact TEV protease recognition site reduceskcat/Km by more than an order of magnitude for pep-tide-based substrates [22]. This measurement performedwith intact protein substrates shows that Lys is not welltolerated by TEV protease in this context also.

Proteolysis of multidomain fusion proteins with TEV

protease

Two A. thaliana genes were expressed in pVP14 andpVP15 to establish whether the anisotropy assay could

Fig. 8. SDS–PAGE analysis of protease substrates incubated withFactor Xa and TEV protease for 16 h. Lane M, molecular weightstandards of 31 and 45 kDa; lanes 1–5, Factor Xa with substrates 21-F,22-F, 23-F, 24-F, and 25-F, respectively; lanes 6–10, trypsin withsubstrates 21-F, 22-F, 23-F, 24-F, and 25-F, respectively; lanes 11–14,TEV protease with substrates 21-F, 22-F, 23-F, and 24-F, respectively.

teasesa

F 24F 25F 26F

.1 0.4 0.5 n.d.c

0.2 n.d. 1.01 1 0.05 0.0.02 0.0 1 0.0.02 -0.01 0.0 1

with the indicated combination of protease and substrate. The relativen of a protease with the preferred cognate substrate. For example, theed with substrate 22-F.


be used more generally to monitor removal of fusiontags using TEV protease. Fig. 9A shows time-dependentdecreases in anisotropy for both of the Arabidopsis pro-tein fusions tested that were similar to that observed forreaction of the His6-Cys4-X-MBP substrates. The differ-ence in starting mr values shown in Figs. 9A and B re-flect differences in the molecular masses of thesubstrates created by the pVP14 (18.7 kDa; His6-Cys4-TEV site-At3g03410) and pVP15 (72.9 kDa; His6-MBP-TEV site-His6-Cys4-TEV site-At3g16990) vectors

Fig. 9. Comparison of proteolysis of Arabidopsis proteins expressedfrom either pVP14 or pVP15. The fluorescence anisotropy assays wereconducted as described under Materials and methods. (A) Reaction of5.0 lM At3g03410 expressed from pVP14. (B) Reaction of 5.0 lMAt3g16990 expressed from pVP15. Control reactions (�) contained noprotease; proteolysis reactions contained 0.5 lM TEV protease (m).For one data point in (A) and (B) the symbol was omitted to reveal theerror bar.

and conformational flexibility of the N-terminal versusinternal location of the tetraCys motif.

Fig. 9B shows anisotropy changes for proteolysis of afusion protein produced from pVP15. This fusion pro-tein has the schematic structure shown in Fig. 2B, wherethe tetraCys motif is located between the MBP and theArabidopsis target protein and is flanked on both sidesby TEV protease sites. Reaction at both protease sitesis required to liberate the �3-kDa tetraCys motif, whichby virtue of its small constant size provides a constantlow anisotropy that is independent of the compositionof the original large and variable-sized fusion protein.Thus pVP15 provides a context for studies of proteolyticprocessing of fusion proteins fundamentally differentfrom that of the N-terminal modification provided bypVP14.

As with proteolysis from pVP14, a time-dependentdecrease in anisotropy was observed for proteolysis ofArabidopsis targets from the pVP15-derived fusion pro-tein. Since two protease cleavage sites were present inthese fusion proteins, proteolysis reactions could poten-tially contain four different fluorescent species. These in-clude the intact fusion protein, a labeled target proteinarising from proteolysis at the site on the N-terminalside of the fluorophore, a labeled MBP arising from pro-teolysis at the site on the C-terminal of the fluorophore,and the labeled tetraCys motif arising from proteolysisat both sites. Since each of these species could poten-tially exhibit a unique anisotropy, it is notable that theoverall change in anisotropy was well fit as a singleexponential. Multiple reaction schemes could accountfor this behavior, and making use of different proteasesin the pV15 construct can test these possibilities.

Discussion

Fluorescence anisotropy has been used to monitorproteolysis reactions, but these previous efforts werelimited by the use of conventional nonspecific fluoro-phore labeling reagents, which gave heterogeneous sub-strates containing multiple labels. The development ofbis-arsenical fluorophores has made it possible to fluor-escently label proteins at a specific position by intro-duction of the tetraCys motif [14]. Specific proteinlabeling opens up the possibility for many in vivoand in vitro applications that are not possible usingtraditional fluorophore labeling reagents [23,24]. Thepossibility of using fluorescence anisotropy to providetime-resolved detection of protease reactions has beenthe focus of this work.

Assay advantages

After in vitro introduction of site-specific proteinlabeling, the assay method reported here enables real-


time monitoring of proteolysis. The advantages com-pared to other proteolytic assays are most apparentfor site-specific proteases when it is desirable to monitorthe cleavage of a single peptide bond. By making use ofmodel substrates containing a tetraCys motif and E. coli

MBP, the activity of different proteases can be studiedafter modifying the linker peptide between the tetraCysmotif and the MBP to contain the protease recognitionsite of interest. A comparison of proteolysis extent deter-mined using this anisotropy-based technique with thewidely accepted method of SDS–PAGE gave a highlyfavorable correlation, as shown in Fig. 6. In addition,initial velocity data for proteolysis were linear with re-spect to the concentration of all proteases tested withinlimits imposed by instrumentation and fundamentalconditions of the methods development. Taken together,the results demonstrate that the fluorescence anisotropycan indeed be applied to monitor specific proteaseactivity.

Assay limitations

During the labeling reaction, other Cys-rich host pro-teins may occasionally react with the bis-arsenical flu-orophores [25]. In E. coli, SlyD is the most abundanthost protein that is typically labeled [26]. For the major-ity of circumstances, nonspecific labeling does not pres-ent a problem for the anisotropy measurement, as thismeasurement is dependent on reaction at a protease sitethat will not likely be present in the labeled host protein.

At high concentration of protease, the initial rates forproteolysis deviated from linearity. For TEV protease,linearity was not maintained for concentrations above2 lM. Since the substrate concentration was 20 lM,the steady state kinetics assumption of the availabilityof a large excess of substrate might no longer be valid.For trypsin, an instrumental limitation was likelyresponsible for nonlinearity as the time required to ob-tain the individual anisotropy time point measurementsapproached the time constant of the proteolysis reac-tion. As implemented, anisotropy measurements takenwith the multiwell plate reader require two measure-ments: one parallel to excitation polarization and oneperpendicular. Overall, this process required a minimumof 24 s to complete for a single sample and as long as7 min to complete when 384 samples were measured inparallel. For most of the results reported here, the pro-tease reactions took place over a sufficiently long timescale that compensation for the delay between the mix-ing and the first measurement was not necessary. How-ever, the large changes in anisotropy that occurred inshort time periods with the higher concentrations oftrypsin (trypsin is also the protease with the highestturnover number; see Fig. 5) resulted in measurementinaccuracies that may have affected the initial rate deter-mination. If necessary, optimization of the concentra-

tion of protease or the number of samples underinvestigation could help to avoid this problem inpractice.

Comparison of reaction rates

The turnover number determined for trypsin was 3-fold greater than thrombin and 10-fold greater thanTEV protease (see Fig. 5A) for reaction with their cog-nate His6-Cys4-X-MBP substrates. The lower reactionrate for TEV protease may be a result of the stringentsubstrate specificity for this enzyme, which demandsan extended substrate–protein interaction for catalysis[27]. When compared on a unit activity basis, FactorXa, enterokinase, and thrombin had specific reactionrates within a factor of three for proteolysis of their cog-nate His6-Cys4-X-MBP substrates. Although not large,this variation may be due to intrinsic catalytic propertiesof the different enzymes, differences in the protein sub-strates used by the manufacturer to determine the re-ported protease activity, reaction conditions used forthe anisotropy measurements, minor variations in theHis6-Cys4-X-MPB substrates introduced by the changesin protease recognition site, or other unknown factors.

Measurement of nonspecific proteolysis

A consequence of the recombination-based cloningwas that all substrates contained two Lys residues inthe linker peptide between the tetraCys motif and theMBP, allowing trypsin to react at these positions. Notsurprisingly, all of the model substrates were susceptibleto trypsin proteolysis. MBP was resistant to trypsin ex-cept for a small amount of proteolysis at a surface-ex-posed loop near the C-terminal (Arg354 in PDB entry1ANF [28]), which was detected only after extendedincubation. Release of this small C-terminal peptidefrom the larger fusion protein would not give a changein mass sufficiently large to be reliably detected by theanisotropy measurements. Consequently, the anisotropychange observed with trypsin proteolysis can be attrib-uted to hydrolysis occurring in the linker peptide be-tween the tetraCys motif and the MBP. Although theinitial reaction rates were linear with respect to trypsinconcentration with the substrates tested here, the pres-ence of multiple trypsin-susceptible sites in other fusionproteins could potentially complicate efforts to relate theassay data to fundamental kinetic parameters.

Enterokinase and Factor Xa exhibit nonspecific cleavage

The proteases investigated here are commonly usedfor the removal of fusion tags from recombinant pro-teins. Among these, TEV protease and thrombin provedto have the highest fidelity for substrate recognition inthis study. For TEV protease, significant proteolysis oc-


curred only for substrate 26-F, which was engineered tocontain the appropriate recognition site.

Thrombin reacted with protease substrate 25-F, engi-neered for thrombin susceptibility, but also reacted with22-F, which did not contain the accepted thrombin rec-ognition sequence. Matrix-assisted laser desorption ion-ization MS of reaction mixtures revealed a fragmentconsistent with proteolysis after Arg28 of substrate22-F, consistent with the thrombin P1 specificity. More-over, enterokinase and Factor Xa reacted well with sub-strates that did not contain protease recognition sitesdesigned for those proteases (see Table 3). Nonspecificproteolysis has been reported in the past for these en-zymes [9], which has motivated the use of the more spe-cific viral proteases such as TEV protease for fusionprotein processing. All of the nonspecific proteolysis ob-served with enterokinase and Factor Xa occurred withinthe linker peptide between the tetraCys motif and theMBP.

Application of fluorescence anisotropy to protease

assays in high-throughput protein production

Assays using the His6-Cys4-X-MBP substratesshowed the potential for using fluorescence anisotropyto monitor proteolysis reactions. In practice, this con-struct (see Fig. 2A) would also be suitable for investigat-ing expression and proteolysis of other highly solubleproteins. For less soluble proteins, solubility can be dra-matically enhanced by expression as a fusion proteinwith a solubility domain (MBP, thioredoxin, glutathioneS-transferase, NusA; see Fig. 1B). Assembly of this typeof multidomain fusion protein places the protease site ina structural context different from that of the N-terminalposition. The results of Fig. 9B show that anisotropycan be reliably used to monitor proteolysis from thismultidomain fusion context also.

Conclusions

In this work, we have given a protein cloning strategyand expression vector design that allows production ofprotein fusions incorporating the tetraCys motif in func-tional proximity with protease recognition sites. Uponlabeling of these recombinant proteins with bis-arsenicalfluorophores, proteolysis can be monitored in real timeby the use of fluorescence anisotropy, a technique thatis well suited to high-throughput implementation. Thesensitivity of the fluorescence-based anisotropy assayand the reliability offered by numerical analysis oftime-resolved data suggest additional utility in optimiza-tion of protease reaction conditions, evaluation of alter-native linker peptides in multidomain fusion proteins,and investigations of inhibitors of protease reactions.These are but a few of the ongoing critical problems in

basic research and applications of important proteolysisreactions [9,29].

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