Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding

A. Hrzenjak1, S. Frank1, B. Maderegger2, H. Sterk2 and G.M. Kostner1,3

1 Institute of Medical Biochemistry and Medical Molecular Biology, University of Graz, Harrachgasse 21/III 2 Institute of Organic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB100. In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution 1H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC50 with {epsilon}-ACA 0.47 mM versus 2–11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.

Keywords: apo(a)/1H-NMR/kringle/Lys-Sepharose/refolding


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lipoprotein (a) [Lp(a)] is as a cholesterol-rich, LDL-like particle found in plasma of humans, old-world primates and the European hedgehog. Lp(a) consists of apo(a) that is disulfide linked to apoB100, the main protein component of LDL. Elevated plasma Lp(a) concentration (>30 mg/dl) is recognized as an independent risk factor for cardiovascular diseases in humans (Kostner et al., 1981Go; Utermann, 1989Go, 1990Go; Scanu and Fless, 1990Go). It should be pointed out that neither the physiological function of Lp(a) nor the site and mechanism of its catabolism is known.

Apo(a) is highly glycosylated protein sharing a high degree of sequence homology with plasminogen (McLean et al., 1987Go). This includes the sequences of the KIV domain of plasminogen, which are found in type 1 to type 10 (T1–T10) apo(a) kringles, KV and the protease domain, the latter lacking an apparent proteolytic function in Lp(a). The Lp(a) assembly takes place extracellularly and a two-step model has been proposed (Chiesa et al., 1992Go; Koschinsky et al., 1993Go; White and Lanford, 1994Go; Trieu and McConathy, 1995Go; Frank and Kostner, 1997Go). In the first step, unique kringles bind non-covalently to Lys and possibly other residues of apoB100 from LDL yielding an unstable complex which is dissociable with Lys analogues, proline and other charged molecules (Frank et al., 1995Go). In the second step, the complex is stabilized by disulfide bridge formation between Cys4057 of apo(a) and Cys4326 of apoB100. Previous studies (Frank et al., 1994Go, 1995Go; Trieu and McConathy, 1995Go) ascribed kringle IV-T6 the leading role in the first step of Lp(a) assembly. To study the mechanisms involved in Lp(a) assembly further we cloned, overexpressed and purified apo(a) KIV-T6 using an Escherichia coli expression system.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PCR cloning of KIV-T6

Restriction enzymes were obtained from New England Biolabs, unless specified otherwise. Human apo(a) KIV-T6 was PCR amplified together with its interkringle sequences (amino acids 1115–1227) using human cDNA (McLean et al., 1987Go) as a template. PCR amplification was performed using the following two synthetic oligonucleotides:

To permit the cloning of KIV-T6 sequence into the expression vector, an EcoRI restriction site was included in the N-terminal primer and a HindIII and two STOP codons were included in the C-terminal primer. The His6-tag sequence in the N-terminal primer allows single-step purification of recombinant protein by use of TALON metal affinity chromatography (Clontech); the His-tag may be removed by factor Xa cleavage. The conditions for 50 µl polymerase chain reaction (PCR) volume were as follows: 10 pM of oligonucleotides A and B each, 250 ng dNTP, 0.6 units of Taq-polymerase (Finnzymes) and 50 ng of human apo(a) cDNA as a template. The reaction conditions were as follows: denaturing temperature, 94°C, 60 s; annealing, 60°C, 60 s; extension, 72°C, 90 s; 25 cycles. PCR amplification of the 375 bp product was followed by gel purification, digestion with EcoRI and HindIII and ligation into EcoRI/HindIII cleaved pT7-7 vector. This construct (pT7-7/KIV-T6) was used to transform E.coli DH5-{alpha} competent cells in order to amplify recombinant plasmid. Positive clones, confirmed by restriction analysis and DNA sequencing, were finally transformed into E.coli strain BL-21 (DE3) (Novagen).

Expression of apo(a) KIV-T6

The recombinant protein was expressed in E.coli strain BL-21 (DE3) under the following conditions: 1 l of LB medium [10 g of tryptone (Difco), 5 g of yeast extract (Difco), 5 g of NaCl/l distilled H2O, pH 7.4] containing 50 µg/ml ampicillin (Sigma) was inoculated with 10 ml of overnight culture of BL-21 (DE3) containing the pT7-7/KIV-T6 plasmid and incubated at 37°C with agitation (250 r.p.m.) until the culture achieved an A600 nm of 0.5. At this point the expression of recombinant apo(a) KIV-T6 was induced by addition of 0.05 mM isopropyl-ß-D-thiogalactoside (IPTG) (Sigma) and the culture was incubated for an additional 4 h. During this time, A600 nm reached ~1.0 and ~1.9 for induced and non-induced cultures, respectively. Cells were harvested by centrifugation at 6000 g for 10 min at 4°C and the cell pellets were frozen at –70°C until use. Approximately 2 g of cells were obtained per liter of culture medium. Aliquots from induced and non-induced cells were taken, frozen at –20°C and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), followed by Coomassie Brilliant Blue staining in order to visualize protein bands.

Cell fractionation

After thawing, the cell pellet from 1 l of culture was resuspended in 50 ml of lysis buffer [(20 mM Tris–HCl, 100 mM NaCl, 8 M urea (Sigma), 200 µl of 5 mg/ml DNase I (Sigma), final pH 8.0] and this suspension was gently stirred for 30 min on the turn-wheel (100 r.p.m.) at room temperature. To reduce viscosity, the suspension was additionally passed through A 21-gauge syringe needle and finally centrifuged at 12 000 g for 15 min at 4°C. The supernatant was used for further purification of apo(a) KIV-T6.

Purification of recombinant apo(a) KIV-T6

The purification of KIV-T6 was a combination of batch/gravity flow column. TALON metal affinity chromatography is based on the affinity of TALON for His-tagged recombinant protein (Clontech). TALON resin was thoroughly resuspended, an aliquot (2 ml of resin suspension per 5 mg of anticipated His-tagged protein) was briefly centrifuged at 700 g for 2 min at room temperature to pellet the resin, the supernatant was discarded and the resin was additionally washed with three volumes of lysis buffer as described in the original protocol (Clontech). Cell supernatant after cell fractionation was added to the resin and the suspension was gently stirred for 30 min on the turn-wheel (60 r.p.m.) at room temperature. The sample was centrifuged at 700 g for 5 min at room temperature and the supernatant was discarded. The resin–protein complex was washed twice with 30 ml of lysis buffer, followed by incubation for 10 min at room temperature and centrifugation at 700 g for 5 min each time. The resin–protein complex was transferred into the column (5x50 mm) and washed three more times using 20 ml of wash buffer lacking urea (20 mM Tris–HCl, 100 mM NaCl, 10 mM imidazole, final pH 8.0) in order to remove traces of urea and non-specific proteins. Finally, apo(a) KIV-T6 was eluted with elution buffer (20 mM Tris–HCl, 100 mM NaCl, 50 mM imidazole, 1 mM CaCl2, final pH 7.5) at a flow-rate of 0.5 ml/min. Protein was monitored at 280 nm and 0.5 ml fractions were collected. Proteolytic cleavage of purified His-tagged KIV-T6 was performed immediately after purification (see Results).

To confirm the proper kringle structure after refolding, the KIV-T6 was applied on a Lys-Sepharose 4B column (Pharmacia Biotech) followed by a wash with wash buffer A (50 mM Tris–HCl, pH 8.0) and twice with wash buffer B (50 mM Tris–HCl, 0.5 M NaCl, pH 8.0). Protein was eluted with elution buffer [50 mM Tris–HCl, 0.2 M {varepsilon}-aminocaproic acid ({varepsilon}-ACA), pH 8.0] at a flow-rate of 1 ml/min and 1.0 ml fractions were collected.

Proteolytic cleavage

For removal of the His-tag, recombinant His-tagged KIV-T6 was treated with factor Xa. Cleavage was performed immediately after purification (see Results) in modified cleavage buffer (20 mM Tris–HCl, 100 mM NaCl, 50 mM imidazole, 1 mM CaCl2, final pH 8.0). A ratio of 5 units of factor Xa per milligram of recombinant protein was used and the mixture was gently stirred at 25°C for 16 h. To monitor the cleavage efficiency, the mobility shift of KIV-T6 was followed by SDS–PAGE and immunoblotting.

SDS–PAGE and immunoblot analysis

Aliquots of cell lysates and fractions containing KIV-T6 were mixed with sample buffer [20% (w/v) glycerol, 5% (w/v) SDS, 0.15% (w/v) bromophenol blue, 63 mmol/l Tris–HCl, pH 6.8] and incubated for 10 min at 95°C. Samples were analyzed by SDS–PAGE (15%) for 1 h at 150 V (running buffer: 25 mM Tris, 0.2 M glycine, 3.5 mM SDS) and protein bands were visualized by Coomassie Brilliant Blue staining for 30 min. In some cases samples were transferred to nitrocellulose membranes and incubated with a specific antibody against KIV-T6 (see below). Bands were visualized by enhanced chemiluminescence assay (ECL, Amersham) following the original protocol.

The specific polyclonal antibody against KIV-T6 was prepared by immunizing rabbits with three 500 µg portions of KIV-T6 for 3 weeks. A 50 ml volume of blood was then collected and centrifuged at 1500 g for 15 min at room temperature and plasma was treated with 600 mg/l of CaCl2 to eliminate fibrin. After 1 h of incubation at room temperature the sample was centrifuged at 45 000 g for 10 min; the supernatant was collected, supplemented with 1 mg/ml EDTA and 1 mg/ml NaN3 and stored at –20°C in small aliquots. An antiserum dilution of 1:500 was found to be optimal for immunoblotting.

Refolding of KIV-T6

This was performed as described (Buchner and Rudolph, 1991Go; LoGrasso et al., 1994Go). After exchange of elution buffer by ultrafiltration (Amicon membrane, YM3 = 3000 MW), recombinant protein from 1 l of E.coli culture was resuspended in 3 ml of buffer A [100 mM Tris–HCl, 6 M guanidine–HCl (Sigma), 1 mM EDTA, 0.1 M DTT (Sigma), final pH 8.0] and stirred (40 r.p.m.) at room temperature for 2 h. This solution was diluted with buffer B [100 mM Tris–HCl, 0.4 M arginine (Sigma), 1 mM EDTA, 4 mM oxidized glutathione (Sigma), final pH 8.0] to 100 ml and incubated at 10°C for 5 days. Samples were stored at –20°C until further use.

Reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis

RP-HPLC was carried out on a Hewlett-Packard Series 1050 liquid chromatograph equipped with a 125x4 mm Vydac RP-4 column. A water–acetonitrile gradient, with acetonitrile concentration increasing from 10 to 90% in 60 min, was applied at a flow-rate of 1 ml/min. Both gradient compounds were supplemented with 0.1% trifluoroacetic acid (TFA). The UV absorbance was measured at 215 nm.

1H-NMR spectroscopy

One-dimensional 1H-NMR spectra were recorded at 27°C, using a Varian 600 MHz Inova Unity spectrometer. The NMR sample contained 0.1 mM of human apo(a) KIV-T6 in 15 mM sodium phosphate buffer, pH 7.4, supplemented with 5% D2O. Chemical shifts are reported in p.p.m. relative to the HOD resonance.

125I labeling of KIV-T6

After purification and refolding, 2 mg of KIV-T6 were dialyzed against PBS (phosphate-buffered saline, pH 7.4) and finally incubated with 1.0 mCi of 125I for 5 min at room temperature. In order to separate labeled protein from free 125I, the sample was chromatographed on a Sephadex G25M column followed by dialysis against 15 l of PBS for 14 h at 4°C. The final protein concentration as determined by the Lowry method for the particular batch was 1.82 mg/ml. The specific activity measured in a {gamma}-counter (Cobra Quantum–Packard) was 238 c.p.m./ng protein. The amount of free 125I in the sample was 5.4%.

Lys binding properties of refolded KIV-T6

125I-labeled KIV-T6 was incubated for 1 h with 2 ml of Lys-Sepharose at room temperature. After filling into a small column, the Lys-Sepharose was washed once with 4 ml of PBS, twice with 4 ml of 40 mM NaCl and additionally twice with 4 ml of PBS in order to remove NaCl. Elution was performed with increasing concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.8 and 2.2 mM) of {varepsilon}-ACA. Fractions of 4 ml were collected and the protein concentrations were determined by measuring the radioactivity.

In order to study the affinity for LDL, refolded KIV-T6 was subjected to competition experiments for Lp(a) assembly. LDL was purified from healthy volunteers as described previously (Frank and Kostner, 1997Go). Amounts of 2 µg of human LDL were incubated with the recombinant apo(a) `XL' (Frank et al., 1994Go) expressed in transiently transfected COS-7 with the admixture of various ratios of refolded KIV-T6 in DMEM medium, for 16 h at 37°C. The total reaction volume was 300 µl. The amount of apo(a) XL complexed to LDL (assembly rate) was determined by a DELFIA immunoassay, as described previously (Frank et al., 1999Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PCR cloning and expression of apo(a) KIV-T6

The cDNA for recombinant KIV-T6 was engineered by PCR in such a way as to contain two specific restriction sites (EcoRI/HindIII) for insertion into the E.coli expression vector. The steps involved in this procedure are summarized in Figure 1Go. Additionally, a His6-tag and an Xa-cleavage site were added to the N-terminus to simplify purification of recombinant protein and the possibility of proteolytic removal of the His6-tag. The remaining three amino acids which precede the first histidine encoded by pT7-7 bacterial vector are Met–Ala–Arg. The calculated molecular weight of the whole recombinant protein was 15.9 kDa.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Cloning strategy and construction of the expression vector for KIV-T6. Vector pT7-7 apo(a) KIV-T6 contains a T7 promoter, a 5'-polyhistidine tag, Xa restriction site followed by 318 bp coding sequence of apo(a) KIV-T6 and two stop codons.

 
The recombinant human apo(a) KIV-T6 cDNA inserted into the pT7-7 vector was amplified in DH5-{alpha} E.coli. Several clones were screened by restriction analysis and positive recombinant vectors were transformed into BL-21 (DE3) E.coli which are inducible with IPTG. Four different IPTG concentrations (0.4, 0.1, 0.05 and 0.02 mM) were tested; results are presented in Figure 2AGo. The optimal IPTG concentration was found to be 0.05 mM and was used for induction during further time-course and scale-up experiments. The time-course experiment for expression of apo(a) KIV-T6 is presented in Figure 2BGo. BL-21 (DE3) cells which had not been induced with IPTG showed no detectable expression of the 16 kDa apo(a) KIV-T6 protein band. After induction with IPTG, cells were harvested at different time points and total bacterial proteins were fractionated by SDS–PAGE (15%) followed by Coomassie Brilliant Blue staining. As shown in Figure 2A and BGo, the pattern of total bacterial protein changed significantly after induction. A time-dependent increase in expression of 16 kDa recombinant protein was observed. The best relation between recombinant protein and total cell proteins was observed 4 h after induction.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. Induction experiments and time course for apo(a) KIV-T6. (A) Cells were induced at A600 nm = 0.5 with different IPTG concentrations. Four hours after induction the cell pellet was lysed and analyzed by 15% SDS–PAGE. Lane 1 contains cell lysate from non-induced cells. Lanes 2–5 contain cell lysates from cells induced with 0.4, 0.1, 0.05 and 0.02 mM IPTG, respectively. (B) BL-21 (DE3) E.coli cells containing the pT7-7/KIV-T6 construct were induced at A600 nm = 0.5 with 0.05 mM IPTG. Aliquots of 1 ml were taken at the defined time points and centrifuged and cell pellets were lysed with SDS loading buffer. An equal mass of protein was applied to each lane. Electrophoresis was carried out using 15% SDS–PAGE and visualized by Coomassie Brilliant Blue staining. Lanes 1–7 contain aliquots of cell lysates induced for 0, 1, 2, 3, 4, 6 and 7 h, respectively. Lane 8 contains lysozyme as a molecular marker of 14 kDa; the 16 kDa band of KIV-T6 is marked with an arrow.

 
Purification of His-tagged apo(a) KIV-T6

We examined various fractions of E.coli cells for the presence of KIV-T6 in order to determine the subcellular distribution of recombinant protein (data not shown). As reported previously for plasminogen and for other apo(a) kringles (Cleary et al., 1989Go; LoGrasso et al., 1994Go; Chung et al., 1998Go), most of the expressed KIV-T6 was present as insoluble protein in inclusion bodies. To obtain pure KIV-T6, inclusion bodies were solubilized with lysis buffer containing 8 M urea (see Materials and methods) and recombinant apo(a) KIV-T6 was isolated using TALON metal affinity chromatography. All proteins lacking His6-tag were eluted in the few wash steps, whereas apo(a) KIV-T6 remained bound to the column until elution with imidazole-containing elution buffer (Figure 3AGo). Figure 3BGo shows that the scale-up isolation in a one-step purification is very efficient, resulting in high purity (>95%) of human apo(a) KIV-T6. The solubility of pure KIV-T6 in Tris–HCl, pH 8.0, was >10 mg/ml. In 10 mM HEPES buffer, pH 7.0, the solubility decreased to ~8 mg/ml.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Purification of recombinant apo(a) KIV-T6. (A) After induction for 4 h, cells were lysed with lysis buffer, incubated with TALON metal affinity resin, washed three times with wash buffer and eluted using imidazole elution buffer (for details, see Materials and methods). 15% SDS–PAGE of different fractions: 1 = non-induced cells; 2 = induced cells; st = 14 kDa lysozyme standard. (B) Elution profile from 1 l of culture medium. Protein was eluted with 100 µl portions of elution buffer; protein concentrations were determined by the Lowry method. The level of expression in this experiment was >7 mg/l of culture medium.

 
Proteolytic cleavage of recombinant protein

In order to remove N-terminal His6-tag from KIV-T6, proteolytic cleavage was performed with factor Xa. This protease recognizes the Ile–Glu–Gly–Arg tetrapeptide that was cloned between the His6-tag and the KIV-T6 sequences. We noticed that in our case the proteolytic cleavage was inefficient using cleavage buffer recommended by Pharmacia Biotech (50 mM Tris–HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5). This was the case especially for the fractions which were stored at –20°C for more than 48 h and we presumed that this is caused by masking of the Xa cleavage site due to formation of intra- or inter-kringle accidental disulfide bonds during the protein storage. In order to avoid this, we prepared cleavage immediately after purification through the TALON column. After loading, the column was washed with buffer containing 8 M urea in order to remove non-specific proteins. Three additional wash steps followed using wash buffer without urea in order to remove traces of urea from the column. Finally, the cleavage was performed immediately after elution in the elution buffer with addition of 1 mM CaCl2 and factor Xa in the absence of urea. These modifications of cleavage procedure were necessary to increase the cleavage efficiency and they enabled us to obtain His-tag free KIV-T6 with a yield of ~70%. The cleavage efficiency was determined by SDS–PAGE and immunoblotting, yielding a shift in electrophoretic mobility which corresponds to a molecular size shift of ~1.8 kDa (Figure 4Go).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. Factor Xa-protease digestion of His-tagged recombinant apo(a) KIV-T6. Purified KIV-T6 was digested with factor Xa as described under Materials and methods. After 16 h of digestion cleaved KIV-T6 (~70%) was separated by TALON affinity chromatography from non-digested protein and subjected to 15% SDS–PAGE followed by immunoblotting using a specific antiserum against KIV-T6. 1 = KIV-T6 before cleavage; 2 and 3 = two different concentrations of KIV-T6 after Xa-protease cleavage and separation by TALON affinity chromatography.

 
Refolding of recombinant KIV-T6

As the purification of recombinant apo(a) KIV-T6 from E.coli requires denaturing conditions, refolding was necessary to obtain a native kringle structure. As measures of proper kringle folding, four different methods were applied: binding to Lys-Sepharose (Figure 5AGo), SDS–PAGE, RP-HPLC and 1H-NMR spectroscopy. Figure 5BGo shows the SDS–PAGE pattern of the identical sample once under reducing and once under non-reducing conditions; the non-reduced fraction migrated faster than the reduced fraction owing to its compact kringle structure after refolding.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 5. Binding of refolded apo(a) KIV-T6 to Lys-Sepharose. (A) Refolded KIV-T6 was incubated with Lys-Sepharose for 2 h at room temperature, washed twice with wash buffer A, twice with wash buffer B and then eluted with elution buffer as described under Materials and methods. Volumes of 2 µl of the eluted material were analyzed by 15% SDS–PAGE (line 1). st = low molecular weight standard. (B) Refolded KIV-T6 was analyzed by SDS–PAGE (15%) under reduced (lines 2 and 3: 10 and 20 µg, respectively) and non-reduced conditions (line 4).

 
RP-HPLC was applied to detect whether major fractions of unfolded or misfolded protein, which would cause different retention behavior on an RP column, are present in the purified protein. A sample of 150 µg of purified apo(a) KIV-T6 was applied to the column. As displayed in Figure 6Go, >99% of the protein eluted from HPLC column as one sharp and symmetrical peak. A minor fraction comprising 0.89% was also observed.



View larger version (7K):
[in this window]
[in a new window]
 
Fig. 6. Elution of KIV-T6 from RP-HPLC: 150 µg of purified and refolded apo(a) KIV-T6 were applied to a Vydac RP-4 column (Hewlett-Packard) and eluted with a water–acetonitrile gradient (see Materials and methods). The y-axis displays the absorbance at 215 nm in milli-arbitrary units. The major peak comprised 99.11%. In addition, a small impurity comprising 0.89% of the peak area was also detected.

 
1H-NMR analysis of refolded protein

Purified refolded human apo(a) KIV-T6 was investigated by 1H-NMR spectroscopy to verify the correct folding. Several investigators have demonstrated in the past the appearance of proton resonances at ~0.2 and –1.0 p.p.m., which were considered as fingerprints for a proper kringle folding (DeMarco et al., 1982Go; Trexler et al., 1983Go; Ramesh et al., 1987Go; Thewes et al., 1987Go). These resonances arise from the {delta},{delta}1-CH3 protons of the conserved Leu46 residue by interaction with several ring currents caused by aromatic amino acid residues (DeMarco et al., 1985Go). This fact has also been used previously to provide evidence for a proper refolding of recombinant human plasminogen kringle I (Menhart et al., 1991Go) and human apolipoprotein(a) kringle 37 (LoGrasso et al., 1994Go). The high-field region of a one-dimensional 1H-NMR spectrum is shown in Figure 7Go. Resonances observed at 0.20, 0.15 and –1.05 p.p.m., respectively, in our case also demonstrated a proper structure of the refolded apo(a) KIV-T6.



View larger version (66K):
[in this window]
[in a new window]
 
Fig. 7. High-field 1H-NMR spectrum of refolded apo(a) KIV-T6. The 1H-NMR spectrum was measured at 600 MHz at 25°C. The final KIV-T6 concentration in 15 mM sodium phosphate buffer, pH 7.4 (5% 2H2O), was 0.1 mM. Arrows indicate characteristic kringle signals.

 
Binding of 125I-labeled apo(a) KIV-T6 to Lys-Sepharose

In order to study the binding affinity of KIV-T6 to surface-bound Lys, 125I-labeled protein in PBS, pH 7.4, was incubated with Lys-Sepharose and specifically bound material was eluted with increasing concentrations of {varepsilon}-ACA ranging from 0.2 to 2.2 mM. By semilogarithmically plotting the fraction of bound KIV-T6 versus the {varepsilon}-ACA concentration, an EC50 value of 0.47 mM was obtained (Figure 8Go).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8. Determination of the binding affinity of KIV-T6 to Lys-Sepharose. Refolded KIV-T6 was bound to Lys-Sepharose and eluted with increasing concentrations of {varepsilon}-ACA. By extrapolation of 50% binding to the x-axis, an EC50 value of 0.47 mM was found.

 
Inhibition of Lp(a) assembly by KIV-T6

In order to investigate the extent to which refolded KIV-T6 might be able to interfere with the Lp(a) assembly using LDL and an intact recombinant apo(a) containing 18 KIV repeats, KV and the protease domain, apo(a)-XL was incubated with LDL in the absence and presence of increasing amounts of KIV-T6. As shown in Figure 9Go, a 100-fold molar excess of KIV-T6 inhibited Lp(a) assembly by only ~20%. At a 1000-fold molar excess the inhibition was not more than 60% compared with the assembly in the absence of KIV-T6.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 9. KIV-T6 inhibition of Lp(a) assembly. Purified human LDL (2 µg) was incubated with recombinant apo(a)-XL (containing 18 KIV repeats as described by Frank et al., 1994) in the presence of refolded KIV-T6 at molar ratios of 1:1 to 1:1000 in 300 µl of DMEM medium for 16 h at 37°C. The rate of assembly was determined by DELFIA as described by Frank et al. (1999). The values are means of three measurements.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of the present study demonstrate that human apo(a) KIV-T6 can be expressed in the E.coli system, purified and properly refolded in vitro. Overexpression of KIV-T6 as a His6-tagged protein in E.coli could be achieved under control of the T7 promoter. The kind of promoter seems to play a crucial role for high-rate expression since first experiments carried out using the pMS470 vector containing the tac promoter, which is known as a very strong promoter for bacterial expression, yielded only very small amounts of KIV-T6 (data not shown). This seems to be a general phenomenon for expressing kringles in bacterial systems as an identical observation was made previously by LoGrasso et al. (1994), who expressed the apo(a) kringle T10. The amount of expressed protein under control of the T7 promoter was generally high. After purification and refolding amounts in the region of 7 mg of KIV-T6 per liter of culture medium were obtained. This is 10–15 times higher than the amount of tissue plasminogen activator (t-PA) K-2 expressed (Cleary et al., 1989Go). Other groups (Menhart et al., 1991Go; Li et al., 1992Go) reported on the expression of different plasminogen kringles in E.coli achieving up to ~200 µg/l culture medium. These experiments were performed under control of the tac promoter or the alkaline phosphate (AP) promoter, which appear to be less suitable for the expression of apo(a) kringle domains. However, the possibility that the KIV-T6 cDNA sequence could also play a very important role for the expression still exists and cannot be excluded.

The homogeneity of refolded and purified apo(a) KIV-T6 was assessed by RP-HPLC, showing a single peak and >99% purity. Furthermore, our purified and refolded KIV-T6 was studied by 1H-NMR spectroscopy and exhibited methyl doublet signals at 0.2 and –1.05 p.p.m., an important measure of proper kringle folding (DeMarco et al., 1982Go, 1985Go; Ramesh et al., 1987Go; Thewes et al., 1987Go). These signals arise from the protons of the {delta},{delta}1-CH3 groups of Leu45 and are a strong argument for the integrity of the folding of KIV-T6. This was further confirmed by the demonstration that purified and refolded apo(a) KIV-T6 bind to Lys-Sepharose. However, Lys-Sepharose binding of KIV-T6 was only approximately four times weaker than that of the previously described recombinant apo(a) showing the weakest lysine binding affinity (Frank and Kostner, 1997Go). In that study we observed EC50 values for Lys-Sepharose binding in the range 2–11 mM {varepsilon}-ACA under conditions where KIV-T6 had an EC50 value of 0.47 mM. This is in line with previous observations that the binding affinity of recombinant apo(a) correlates positively with the number of kringles with lysine binding affinity (Frank and Kostner, 1997Go). Our original intention was to investigate the interaction of KIV-T6 with LDL and to map the binding to surface structures of apoB100 in order to obtain further information on the mode of Lp(a) assembly. However, the binding affinity of this single kringle to LDL under physiological conditions was very low (Kd > 10–3 M) (data not shown). In competition experiments using LDL and intact recombinant apo(a) the relatively low affinity of the single kringle KIV-T6 was further underlined: At a 1000-fold molar excess this kringle inhibited only about 60% of the Lp(a) assembly. These findings suggest that the first step of Lp(a) assembly is more complex than simple interaction of apo(a) with Lys residues on the LDL surface. In line with this assumption is the fact that Lp(a) assembly is inhibited not only with Lys analogues but also with Pro and hydroxy-Pro (McConathy and Trieu, 1991Go; Frank and Kostner, 1997Go).

The availability of large amounts of pure native apo(a) KIV-T6 should enable us to perform further functional assembly studies. In addition, KIV-T6 is currently crystallized and its structure will be studied by X-ray crystallography.


    Notes
 
3 To whom correspondence should be addressed.E-mail: gerhard.kostner{at}kfunigraz.ac.at Back


    Acknowledgments
 
This work was supported by grants from the Austrian Research Foundation (FWF-P11691, SFB F702) and the Austrian National Bank (ÖNB 7475). HPLC analysis was performed at PI-CEM, Graz, Austria. The technical assistance of Harald Grillhofer and Anton Ibovnik is appreciated.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Buchner,J. and Rudolph,R. (1991) Bio/Technology, 9, 157–162.[Medline]

Chiesa,G., Hobbs,H.H., Koschinsky,M.L., Lawn,R.M., Maika,S.D. and Hammer,R.E. (1992) J. Biol. Chem., 267, 24369–24374.[Abstract/Free Full Text]

Chung,F.Z., Wu,L.H., Lee,H.T., Mueller,W.T., Spahr,M.A., Eaton,S.R., Tian,Y., Settimi,P.D., Oxender,D.L. and Ramharack,R. (1998) Protein Express. Purif., 13, 222–228.[ISI][Medline]

Cleary,S., Mulkerrin,M.G. and Kelley,R.F. (1989) Biochemistry, 28, 1884–1891.[ISI][Medline]

DeMarco,A., Hochschwender,S.M., Laursen,R.A. and Llinas,M. (1982) J. Biol. Chem., 257, 12716–12721.[Free Full Text]

DeMarco,A., Laursen,R.A. and Llinas,M. (1985) Biochim. Biophys. Acta, 827, 369–380.[ISI][Medline]

Frank,S. and Kostner,G.M. (1997) Protein Eng., 3, 291–298.

Frank,S., Durovic,S. and Kostner,G.M. (1994) Biochem. J., 304, 27–30.[ISI][Medline]

Frank,S., Durovic,S., Kostner,K. and Kostner,G.M. (1995) Arterioscl. Thromb. Vasc. Biol., 15, 1774–1780.[Abstract/Free Full Text]

Frank,S., Hrzenjak,A., Kostner,K., Sattler,W. and Kostner,G.M. (1999) Biochim. Biophys. Acta, 1438, 99–110.[ISI][Medline]

Koschinsky,M.L., Coté,G.P., Gabel,B. and van der Hoek,Y.Y. (1993) J. Biol. Chem., 268, 19819–19825.[Abstract/Free Full Text]

Kostner,G.M., Avogaro,P., Cazzolato,G., Marth,E. and Bittolobon,G. (1981) Atherosclerosis, 38, 51–61.[ISI][Medline]

Li,Z., Gambino,R., Fless,G.M., Copeland,R.A., Halfpenny,A.J. and Scanu,A.M. (1992) Protein Express. Purif., 3, 212–222.[ISI][Medline]

LoGrasso,P.V., Cornell-Kennon,S. and Boettcher,B.J. (1994) Biol.Chem., 34, 21820–21827.

McConathy,W.J. and Trieu,V.N. (1991) Prog. Lipid Res., 30, 195–203.[ISI][Medline]

McLean,J.W., Tomlinson,J.E., Kuang,W.J., Eaton,D.L., Chen,E.Y., Fless,G.M., Scanu,A.M. and Lawn,R.M. (1987) Nature, 330, 132–127.[ISI][Medline]

Menhart,N., Sehl,L.C., Kelley,R.F. and Castellino,F.J. (1991) Biochemistry, 30, 1948–1957.[ISI][Medline]

Ramesh,V., Petros,A.M., Llinas,M., Tulinsky,A. and Park,C.H. (1987) J. Mol. Biol., 198, 481–498.[ISI][Medline]

Scanu,A.M. and Fless,G.M. (1990) J. Clin. Invest., 85, 1709–1715.[ISI][Medline]

Thewes,T., Ramesh,V., Simplaceanu,E.L. and Llinas,M. (1987) Biochim. Biophys. Acta, 912, 254–269.[ISI][Medline]

Trexler,M., Banyai,L., Patthy,L., Pluck,N.D. and Williams,R.J.P. (1983) FEBS Lett., 2, 311–318.

Trieu,V.N. and McConathy,W.J. (1995) J. Biol. Chem., 26, 15471–15474.

Utermann,G. (1989) Science, 246, 904–910.[ISI][Medline]

Utermann,G. (1990) Curr. Opin. Lipidol., 1, 404–410.

White,A.L. and Lanford,R.E. (1994) J.Biol.Chem., 269, 28716–28723.[Abstract/Free Full Text]

Received February 11, 2000; revised July 24, 2000; accepted August 10, 2000.