Design of Helical Proteins for Real-Time Endoprotease Assays

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Analytical Biochemistry 286, 26–34 (2000)doi:10.1006/abio.2000.4780, available online at http://www.idealibrary.com on

Design of Helical Proteins for Real-TimeEndoprotease Assays

Peter Steinrucke,1 Ulrich Aldinger,2 Oliver Hill,3 Alexander Hillisch,4

Renata Basch, and Stephan DiekmannMolecular Biology Department, Institute of Molecular Biotechnology, IMB Jena e.V.,Beutenbergstrasse 11, 07745 Jena, Germany

Received March 20, 2000

Proteases play a key role in cellular biology andhave become priority targets for new pharmaceuti-cals. Thus, there is a high demand for specific, sensi-tive, and quick assays to monitor the activity of endo-proteases. We designed affinity-tagged helicalproteins with unique protease cleavage sites and thusconstructed universal, molecularly defined, and uni-form substrates for in vitro detection of IgA endopro-tease. The substrate is a 10.5-kDa recombinant helicalprotein with a high-affinity (His)6-tag at the amino-erminal end. Further elements are a unique proteo-ytic recognition site and a C-terminal helical exten-ion, which is cut off by the protease. Proteolyticction can be monitored in real time using surfacelasmon resonance spectroscopy. Femtomole amountsf protease could be reliably and quantitatively de-ected within a few minutes after the start of the re-ction. The detection signal changed linearly with themount of protease and was independent of the ap-lied sample flow rate. The biochip can be reversibly

oaded with the recombinant protease substrate, sohat the SPR assay is well-suited for automation. Byubstituting an HIV protease site for the recognitionite of the IgAse, we also obtained a substrate for theuantitative and sensitive detection of HIV-1 endopro-ease. Our substrate design is thus generally applica-le. © 2000 Academic Press

1 To whom correspondence should be addressed at Institute forMolecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Ger-many. Fax: 149-3641-656225. E-mail: [email protected].

2 Current address: CyBio, Carl Zeiss Strasse 1, 07743 Jena, Ger-many.

3 Current address: Graffinity Pharmaceutical Design GmbH, ImNeuenheimer Feld 515, 69120 Heidelberg, Germany.

4
Current address: EnTec GmbH, Adolf-Reichwein-Strasse 20,07745 Jena, Germany.
26

Key Words: protease assays; surface plasmon reso-nance; BIAcore; HIV protease; IgA protease; His-tag.

Proteases are involved in essential biological pro-cesses like blood clotting, controlled cell death, andtissue differentiation. They catalyze important proteo-lytic steps in tumor invasion or the infection cycle of anumber of pathogenic microorganisms and viruses.This makes proteases a valuable target for new phar-maceuticals (1). Consequently, there is a high demandfor versatile assays for quick and easy detection ofprotease activity.

The spectrum of protease substrates and methods tomeasure their turnover has led to a panoply of testswhose results can rarely be compared among eachother. Often used substrates like serum albumin orcasein carry more than one potential proteolytic cleav-age site for endoproteases of lower specificity (2, 3). Inaddition, the proteolytic steps are only poorly definedon the molecular level. After the first round of digestiona heterogeneous mixture of substrate molecules is ob-tained. This precludes an easy kinetic treatment of theproteolytic process. Furthermore, differences in theamino acid sequence of the cleavage sites might resultin a nonuniform kinetic behavior. In addition stericrestrictions can cause differences even in the reactivityof otherwise identical cleavage sites. Chemicalcrosslinking for covalent immobilization is a furtherpotential source for molecular substrate heterogeneity.Thus, we note the desire for well-designed proteasesubstrates, which allow a quantitative activity assayand the comparison of activity data for different pro-teases.

Several methods (4–7) require dye-labeled proteasesubstrates. Reservations can be made about the non-
proteinogenic nature of such labels, their bulky size,
0003-2697/00 $35.00Copyright © 2000 by Academic Press

All rights of reproduction in any form reserved.

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27HELICAL PROTEINS FOR ENDOPROTEASE ASSAYS

and the sterical problems this might cause. Estab-lished label-free methods use electrophoresis (8),HPLC (9), or mass spectrometry (10) for offline mea-surement of protease activity. Alternatively, surfaceplasmon resonance (SPR)5 (11) allows the real-timeand label-free detection of enzymatic activity, providedthat the substrate turnover is correlated with a suffi-ciently high change of mass. Enzymatic SPR assaysrequire that the substrate is immobilized on the bio-chip surface which is preferably done by a high-affinitymolecular tag. Immobilization by tags is molecularlywell defined and more reproducible than conventionalchemical crosslinking. A higher molecular uniformityis achievable and even more desirable for future bio-analytical tools which follow the trend towardnanoscaled miniaturization (12). If function followsstructure biological active surfaces with a desirablehigher grade of functional homogeneity should becomeavailable for biotechnology in this way. The proteasesubstrates presented in this paper monitor endopro-tease activity in real time within a few minutes. AHis-tag is used for specific binding of the substrate tothe biochip surface. The major part of the substrate iscleaved off and its release monitored online by SPR.Altering the protease cleavage site, but keeping theoverall design, enables the construction of very similarsubstrate molecules for different proteases. As an ex-ample a versatile in vitro SPR assay for endoproteaseactivity measurements of IgA and HIV-1 protease isdescribed.

MATERIALS AND METHODS

Substrate Protein Design

Modeling of a-helical proteins was done on a SiliconGraphics Indigo 2 workstation with the biopolymermodule of the SYBYL 6.4 software package (Tripos).Data of the c-Jun protein (13) were used as a firsttarting point. An amino-terminal (His)6-tag was cho-en for immobilization (see Fig. 1b and Fig. 2). The IgArotease site (PTPPTP) was introduced immediatelyarboxy-terminal to the affinity tag. Two glycine resi-ues flank the IgA protease site, so that a higher mo-ecular flexibility of tag and protease cleavage site cane expected. The overall structure of the substraterotein is that of a helical protein. This ensures aell-exposed extended substrate structure. Trp 60 was

ntroduced for the easy UV detection of the protein.he side chain of Cys 92 allows a defined covalent

abeling with fluorescent dyes. The helical moiety com-rises 78 amino acids forming two helical zipper do-

5 Abbreviations used: SPR, surface plasmon resonance; PBS, phos-phate-buffered saline; TAMRA, tetramethylrhodamine-6-maleinim-
ide; Fc2, flow cell 2; Fc1, flow cell 1; TEAA, triethylammoniumacetate.
mains with a hydrophobic backbone of valine andleucine at positions a and d of the repeating hep-tameric abcdefg sequence. Positions b and c are al-ways used by serine residues. At positions e, f, and gcharged amino acids (glutamate, aspartate, and argi-nine) or serine residues were introduced creating adistinctive charge pattern along the helical axis to pre-vent the protein from self-pairing. Amino acids 53–61were inserted for a slight twist of the hydrophobicsurfaces of both zipper domains against each other toreduce unspecific hydrophobic binding of the protein tosurfaces.

The molecular weight of the IgAse substrate waspredicted to be 10.5 kDa. The molecular architecture ofthe HIV protease substrate was basically identical.Only the protease cleavage site was replaced by ex-change against a cleavage site for HIV protease. Theinsert replacement introduced a stretch of 8 aminoacids of a P4-P499 HIV-1 protease cleavage site(SFNF//PQIT) as described by Poorman et al. (14).

Synthesis of Gene Cassettes

The 315-bp gene cassette was synthesized from the39-end by stepwise PCR amplification of 39-overlappingDNA fragments (Fig. 2a). Synthesis started with hy-bridizing the upstream CB fragment (sense strand)with the downstream BA fragment. Overlaps (see Fig.2a, in bold letters) were used to derive a set of specificnested primers for the PCR amplification of the inter-mediate products. A reverse primer, which covered theEcoRI site, was used as downstream primer in allcases. Unique restriction sites were introduced for fur-ther subcloning. The final fragment was digested withNcoI and EcoRI and inserted into the pRSET5D vector(15). Escherichia coli BL21(DE3) (Novagen) was usedas host strain. The sequence of a correct clone wasverified by DNA sequencing on a Licor 4000 sequencer.For synthesis of an HIV protease susbstrate nucleo-tides 43–97 in the IgAse substrate gene were replacedby 59-C CCG GGA TCC GGT GAC TCT TTC AAC TTCCCG CAG ATC ACC GGA GGT CCG GAG GTG TCGAGC TTG GAG TCT CGT GTA TCT AGC-39. This wasachieved by using accordingly altered HG, GF, and FEoligonucleotides in the gene synthesis procedure.

Expression and Purification of Substrate Proteins

Expression of both synthetic genes with the T-7 pro-motor was efficient but also produced inclusion bodies.Clones were raised in Luria broth (100 mg/ml ampicil-llin; 28°C) without further induction until late loga-rithmic growth. Cells were harvested by centrifugationand disrupted by treatment with liquid nitrogen and amortar. The material (approx 10 g) was resuspended in
70 ml phosphate-buffered saline (PBS), sonified for 10

(94

28 STEINRUCKE ET AL.

min, and centrifuged. Sediment was resuspended at70°C with PBS and centrifuged. The supernatant wasapplied to a 10 ml Ni-NTA column. After washing with100 mM potassium phosphate, 100 mM NaCl, pH 7.5,protein was eluted with the same buffer plus 1 Mimidazol. Minor impurities were removed by anion-exchange chromatography on DEAE or by gel filtrationwith Superdex 75 (Pharmacia, Sweden). The correctmolecular weight was confirmed by electrospray massspectroscopy analysis with a Perkin-Elmer Sciex-APIIII quadrupol mass spectrometer. Isolated protein be-haved uniformly in SDS-PAGE but always ran at anapparent molecular weight of approx 18 kDa (Fig. 2b).This might be due to its impaired SDS binding behav-ior caused by the net negative charge of the evenlydispersed glutamate residues. The overall helical na-ture of the substrate proteins was confirmed at 4°C byCD spectroscopy on a JASCO J-710 polarimeter with a0.1 mg/ml solution of the protein in 50 mM potassiumphosphate, pH 7.5. Protein concentrations were mea-

FIG. 1. (a) Working principle of the surface plasmon resonance (structural features of the protease substrate. The substrate protein(His)6-tag and carries a unique protease cleavage site.

sured photometrically. A solution of 2 mg/ml of the

purified substrate protein showed an absorption of 1.0at 280 nm.

Fluorescent Labeling of IgAse Substrate Protein

Ten milligrams of IgA protease substrate was la-beled at its unique carboxy-terminal cystein side chainusing 2 mg of tetramethylrhodamine-6-maleinimide(TAMRA, Molecular Probes, U.S.A.) at 50°C in thepresence of TCEP as reductant (16). Unreacted fluoro-phore was removed by gel filtration on a PharmaciaHigh Trap desalting column. Pooled protein fractionswere lyophilized, redissolved in IgAse buffer (100 mMpotassium phosphate, 50 mM NaCl, 0.005% (v/v)Tween 20), and kept at 220°C until further use.

SPR Measurements

Surface plasmon resonance analysis was carried outat 37°C on a BIAcore 2000 instrument (Biacore, Swe-

R) assay for in vitro measuring of endoprotease activity. (b) Mainamino acids) can be immobilized uniformly by an amino-terminal

SP

den). NTA chips were loaded with 40 ml 0.5 mM nickel

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sulfate in running buffer (10 mM Hepes, 150 mM NaCl,50 mM EDTA, 0.005% (v/v) Tween 20, pH 7.4) applyinga 20 ml/min flow. Substrate protein was diluted inrunning buffer and applied onto the Ni-NTA sensor-chip flow cell 2 (Fc2) with a 5 ml/min flow until a signalf approx 2000 RU could be recorded. HIV proteaseubstrate was used as negative control for the IgArotease assay and was loaded likewise to flow cell 1Fc1) to account for unspecific binding of the proteaseo the sensorchip matrix.

A 60-ml sample of IgA protease (7.5 ng/ml; MoBiTec,Germany) in test buffer (100 mM potassium phos-phate, 50 mM NaCl, pH 7.5) was directed with 6 ml/minthrough reference and detection channel and the timecourse of the SPR signal was recorded. In control ex-periments we changed the flow rate of the enzymesolution from 1 to 10 ml/min without being able todetect notable differences in the signal decrease. In theabsence of enzyme there was no meaningful dissocia-tion of either the substrate or the reference proteinfrom the sensor surface (Fig. 3a). EDTA (60 ml 0.1 M

DTA, 5 ml/min) was used to remove undigested pro-ein or residual protein moieties from the surface of thehip during each regeneration step. After washing withml 0.1 M NaOH, 1 M NaCl (5 ml/min) the chip was

oaded again with nickel for further use as describedbove.The immobilized protein refers to a protein load of 2

g substrate protein/mm2. With a thickness of 800 nmor the sensing evanescent field and a local steady-tate concentration of 5 ng enzyme/ml estimations al-

ways showed a molar excess of substrate protein overenzyme by at least three orders of magnitude.

Data were analyzed with the Origin 5.0 softwarepackage (Microcal, U.S.A.). The signal of Fc2 was cor-rected for unspecific binding effects by subtracting thesignal from the reference channel Fc1. The slope of thecorrected signal 30–90 s after enzyme injection wasused for further analysis (Fig. 3b). The initial slope ofthe signal decrease was measured for various proteaseconcentrations under identical conditions with thesame Ni-NTA biochip (flow rate 6 ml/min in IgAsebuffer). IgAse activity was detected in solutions of 25ng/ml, 12.5 ng/L, 7.6 ng/ml, 5 ng/ml, and 2.5 ng/ml en-zyme. A linear plot was achieved (see Fig. 4a) showingthat the SPR assay can also be used quantitatively.HIV protease activity was measured with identicalexperimental parameters using the immobilized HIV-1protease substrate in the detection channel Fc2 andthe immobilized IgA protease substrate as negativecontrol for the reference channel Fc1. A dilution seriesof HIV-1 protease (Sigma, Germany) with 6, 3, 1.8, 1.2,and 0.6 ng/ml was applied to demonstrate that the testcan also be used quantitatively for other proteases
than IgA protease (Fig. 4b).
Fluorescence Assay

Fluorescent-labeled IgAse substrate protein was in-cubated in free solution with IgA protease from Mo-BiTec. Solid phase extraction with a Ni-NTA spin col-umn (Qiagen, Germany) was used to remove uncleavedprotease substrate: Spin columns were treated with aseries of washing steps prior to use: (1) 1 ml 0.1 MEDTA, (2) 2 3 1 ml 10 mM triethylammonium acetate(TEAA; pH 7.0), (3) 2 3 0.5 ml nickel citrate buffer (100mM NiSO4, 100 mM citric acid, pH 7.0), (4) 1 3 1 ml 10mM TEAA, and (5) 1 3 1 ml IgAse buffer (100 mMpotassium phosphate, 50 mM NaCl). IgAse buffer(1860 ml) was mixed with 100 ml fluorescent-labeledprotease substrate (1 mg/ml) and preincubated for 15min at 37°C. Forty microliters of IgAse (0.25 mg/ml)was added to start the reaction. Samples (400 ml) weredrawn at t 5 0, 15, 30, 60, and 120 min, added to 40ml 4 M NaCl, and centrifuged through a Ni-NTA col-umn (2 min/2000 rpm in a benchtop centrifuge). Eluatewas diluted twofold and the fluorescence was measuredwith a Shimadzu RF-5301PC spectrofluorophotometer(excitation, 555 nm; emission, 575 nm; slit 10 nm). Theresults are shown in Fig. 5.

RESULTS AND DISCUSSION

Rationale for Substrate Protein Design

We constructed simple protease substrates withunique proteolytic cleavage sites and a tag of suffi-ciently high affinity for immobilization. A label-freesurface plasmon resonance assay was set up duringwhich the proteolytic release of the cleavage productfrom the biochip is detected (Fig. 1a). Larger cleavageproducts, which are released from an SPR biochip, areadvantageous (17). However, there are practical uppersize limits for a substrate protein:

Introducing a unique cleavage site for larger sub-strate proteins becomes more difficult. Uniform immo-bilization and accessible presentation of the substratecleavage site are hampered with increasing substratesize. Sterical limitations set an upper limit for theachievable effective concentration of substrate proteinon the SPR biochip surface. Mass transport limitationswill increase for larger cleavage products. We thereforedesigned a helical substrate protein of moderate size(10.5 kDa, Fig. 2a). The same molecular substrate ar-chitecture was used to detect IgA endoprotease (18),which is a serine protease, and for the detection of thefar-less-specific HIV-1 aspartyl protease. The overallhelical structure creates a defined and extended pro-tein conformation. This allows an easy access of theprotease to the unique cleavage site and keeps theterminal affinity tag well exposed. Less sterical de-mands are expected during the immobilization of a
helical substrate than with a bulkier globular protein.

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To maintain the substrate’s spatial flexibility after im-mobilization, the cleavage site was flanked by glycineresidues. Specific cleavage will release more than 85%of the protein amino acids from the biochip. The resid-ual protein remains fixed to the biochip surface. Forautomation purposes a regeneration of the biochip isdesirable. This was achieved by introducing a His-tag,which allows a reversible and repeatable immobiliza-tion of the substrate protein.

Gene Synthesis and Expression

The helical moiety of the protein was designed bycomputer modeling. It has a repetitive amino acid se-quence, so that the higher redundancy of the proteinsequence minimizes the risk of introducing unwantedcleavage sites by chance. Trp 60 was introduced for UVdetection of the protein. Cys 92 can be used for adefined S-alkylation of the protein. The synthetic genewas synthesized by PCR using oligonucleotides and aset of nested primers. The codon usage was adapted tothe needs of an expression in E. coli (19). A 315-bpDNA fragment (Fig. 2a) comprises a 10.5-kDa protein,which was expressed at high levels and could be puri-fied by metal chelate chromatography with a Ni-NTAcolumn (20) in milligram amounts from a 1 L E. coli

FIG. 2. (a) DNA sequence and amino acid sequence of the IgA pubstrate.

culture (Fig. 2b).

Substrate Suitability in a Homogeneous FluorescenceAssay

To prove the functionality of our substrate we testedits proteolytic cleavage in free solution. The high bind-ing affinity of the (His)6-tag enabled a simple fluores-cence assay. The substrate was rhodamine-labeled andcleaved by IgA protease. Uncleaved protein andcleaved His-tag moieties were removed by solid phaseextraction with a Ni-NTA spin column. The extendedhelical structure of the substrate protein ensured thatthe fluorophore could not interfere with the proteolyticcleavage site. In the absence of protease, control exper-iments detected only negligible fluorescence in the el-uate, so that the binding capacity of the spin columnwas sufficiently high. As shown in Fig. 5, IgA proteaseactivity correlated linearly with the presence of fluo-rescent cleavage product in the eluate.

The solid phase extraction is reliable but requires acareful pretreatment of the Ni-NTA chromatographymaterial to ensure the filter function. When we re-peated the experiments in the presence of Tween 20(see Experimental Protocols) we always observed ahigher fluorescence in the eluate (not shown) indicat-ing a certain degree of unspecific binding of the rho-damin-labeled helical moiety. The minimal required

ase substrate. (b) Expression and purification of the IgA protease
rote
sample volume of the fluorescence assay is in the order

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FIG. 3. (a) Sensorgram of an IgA protease assay with a BIAcore 2000 instrument. A Ni-NTA biochip was (a) loaded with nickelimultaneously in both the sample and the reference channel, (b) then loaded in two separate steps first with reference protein in Fc1 (blueolor) and then with substrate protein in Fc2 (green color), and (c) IgA protease was injected (*) passing both flow cells Fc1 and Fc2 . The SPRignal of the reference channel (Fc1, blue colored) was subtracted from that of the sample channel (Fc2, green colored). The result is aorrected signal (red colored) which shows a linear decrease of the SPR signal after addition of the protease Finally the biochip was washedith EDTA (d) to regenerate a nickel-free matrix (e). (b) Detection of the endoproteolytic activity of IgA protease. IgA protease was injectedith a constant flow of 6 ml/min with a buffer containing 7.5 ng/ml IgAse. Shown are the signal drops of two experiments which were carried

out on the same Ni-NTA biochip. The test signal changes linearly with time. The initial slope of the signal 30–90 s after the start of theenzyme reaction was used for further calculations.

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of a hundred microliters if a microcuvette is used. Alabel-free assay with less sample demand is more pref-erable and avoids the possible pitfalls of labeling. Allfurther experiments were therefore carried out labelfree with His-tag-immobilized substrate on SPR bio-chips.

Online Monitoring of Endoprotease Activity bySurface Plasmon Resonance

We used a BIAcore 2000 instrument equipped withNi-NTA biochip to investigate the suitability of our testprotein as a substrate for the 106-kDa IgA protease.

FIG. 4. (a) Quantitative detection of IgA protease. Shown is theinitial rate of proteolysis expressed as the initial slope in the SPRsignal decrease as shown in b. (b) Quantitative detection of HIV-1protease activity.

The enzyme is secreted by the gram-negative diplococ-

cus Neisseria gonorrhoeae and cleaves human IgA1ntibodies at the hinge region (18).As a reference protein, lacking an IgA protease site,e designed and expressed a helical substrate proteinith a cleavage site for HIV-1 protease. In all otherolecular features the reference protein is identicalith the IgA protease substrate. This protein served asnegative control and was loaded onto the reference

hannel (Fc1) of the biochip. The IgA protease sub-trate was loaded onto the detection channel (Fc2).nder saturating conditions we achieved an SPR sig-al of approx 2000 RU in each case. The referencehannel Fc1 is used to account for unspecific bindingffects. As shown in the sensorgram (Fig. 3a), the neg-tive control protein is not cleaved by IgA protease. Annspecific background signal due to leakage of the testr reference proteins from the Ni-NTA matrix could note observed. This confirmed the high affinity of theHis)6-tags in both proteins. When IgA protease was

applied with a constant flow, a linear decrease of thedetection channel signal (Fc2) could be observed (seeFig. 3a and Fig. 3b). Even unspecific binding of the IgAprotease to the helical substrate moiety is taken intoaccount. After data correction with the reference chan-nel, we used the initial slope of the difference signal forour further investigations (Fig. 3b).

The apparent drop of the SPR signal can be smallerthan anticipated due to mass transport limitationsduring the release of the cleavage products from thebiochip matrix. In this case the signal would decreasedependent on the applied flow rate of the enzyme (21).However, a notable flow-rate-dependent change of the

FIG. 5. Fluorescent labeled helical substrates can be used for aneasy assay. Enzyme (5 mg/ml) was added and samples were drawn at
the indicated time points. Uncleaved substrate was removed by solidphase extraction with a Ni-NTA column.

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signal was never observed under tested flow rates from1 to 10 ml/min (data not shown). The observed flow rateindependence in dissociation experiments over a widerrange of experimental flow conditions (8-fold variation)strongly argues against mass transport limitation ef-fects (22). Hence, the slope of the signal decreaseshould clearly correlate with the concentration of theapplied protease.

In a control experiment we took advantage of theeasy regeneration of the Ni-NTA chip. This enabled usto repeatedly use a single biochip. Thus, the measuringbackground of the biochip matrix was about identicalin all cases, which ensured rather uniform test condi-tions as compared to the situation when a number oftests on different biochips are performed. We testedwhether the SPR signal drop correlates with the en-zyme concentration. A moderate sample flow of 6 ml/min was chosen for all experiments. The result isshown in Fig. 4a for the IgA protease assay. The linearbehavior indicates that our assay can also be usedquantitatively, if the enzyme is properly diluted. Un-der conditions of high enzyme loads the decrease is notlinear, however. This can be explained either by masstransport limitation due to macromolecular crowdingeffects (23) or simply might reflect the fact that forsamples of high enzymatic activity the surface concen-tration of the substrate is no longer high enough for aproper kinetic assay. Up to protease concentrations of10 ng/ml its activity can be measured reliably.

Detection times can be cut to the inital 30 to 60 safter injection of the enzyme sample, so that undermoderate sample flow only a few nanograms of IgAprotease are required for detection. This corresponds tothe detection of femtomolar amounts on the minutetime scale. The biochip is easily regenerated with un-cleaved substrate or reference protein. Baseline prob-lems resulting from irreversible precipitation of pro-tein during regeneration have been discussed recently(24), but were not observed in our assay. The helicalpart of the protein is comparably small in size and iswashed out fast and completely with the sample flow.

We chose a second protease to prove that also otherprotease activity tests can be set up in the same way.Retroviral aspartyl proteases are known for their lackof a clear-cut specificity. We selected the HIV-1 aspar-tyl protease as a model of a protease with low andill-defined specificity. The enzyme is a 22-kDa ho-modimer. As a cleavage site we applied the p6/proteasejunction (SFNF PQIT) of the viral pol protein as de-cribed by Partin et al. (25) and Poorman et al. (14). We

used our helical proteins with switched roles: the HIV-protease substrate was loaded onto the detection chan-nel (Fc2) and the IgA protease substrate served as anegative control in the reference channel (Fc1). Whilethe reference protein was not digested, a linear rela-
tionship between the applied enzyme activity and the
initial slope of the SPR signal was observed (see Fig.4b). We found a linear dependence up to 6 ng/ml HIVprotease. At the lower limit this corresponds to thedetection of picomole amounts of enzyme.

To our knowledge this is for the first time that areal-time endoprotease assay with a label-free andwell-defined substrate protein has been demonstrated.Its outstanding features are that the test can be usedquantitatively and with a high sensitivity, while thedetection time for the assay is in the order of minutesand only small sample volumes are required. Newlydesigned and constructed test proteins were usedwhich were uniformly and reversibly immobilized withan affinity tag. The same biochip can be used repeat-edly (more than 20 times) so that the test can beautomated and optimized under screening conditionswith higher throughput. Reference proteins were ap-plied, which lack the specific cleavage site of the testedprotease. In all other features the reference proteinsand their surrounding matched the conditions in thedetection channel as closely as possible. Furthermore,although different proteases are tested with our SPRassay, in all cases after proteolytic cleavage the leavinggroup is the same helical moiety. This keeps dissocia-tion conditions in the biochip matrix uniform and al-lows the quantitative comparison of activity data evenfrom different enzyme sources.

We could demonstrate the suitability of our approachon two clinically relevant proteases of different molec-ular size and specificity. Less pH-dependent affinitytags (e.g., biotin-tags) can be used to extend the prin-ciple of our assay also to acidic proteases. Methods areavailable to ensure a site-specific uniform biotinylationand a higher reversibility of the biotin/avidin binding(26). Currently, in all other cases the advantages of aHis-tag for immobilization are prevailing. Recent stud-ies have shown that His-tagged proteins can also beused in biochips with tethered membranes carryingNi-NTA phospholipids (27). This leaves much room fora biochip design with planar sensor surfaces ratherthan the usual three-dimensional dextran network,which is regularly employed in the SPR biochips.

ACKNOWLEDGMENTS

The authors thank Manfred Raida from the Institute for PeptideResearch (Hannover, Germany) for his support with ESI/MS mea-surements. This work was funded by the German Ministry for Re-search and Technology (BMBF) as part of the BioRegio Program inthe BioRegio Jena.

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Design of Helical Proteins for Real-Time Endoprotease Assays