Host vector system for high-level expression and purification of recombinant, enzymatically active alanine dehydrogenase of Mycobacterium tuberculosis

Gene 212 (1998) 21–29

Host vector system for high-level expression and purification of recombinant, enzymatically active alanine dehydrogenase of Mycobacterium tuberculosis Bernd Hutter, Mahavir Singh * GBF–German National Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany Received 16 October 1997; received in revised form 20 December 1997; accepted 14 February 1998; Received by W. Martin

Abstract The 40-kDa antigen of M. tuberculosis, which is an alanine dehydrogenase, is a species-specific antigen that is potentially useful for strain identification. Large quantities of the purified protein are required for immunological, as well as for detailed biochemical and structural, characterization. The AlaDH gene was cloned by PCR from H37Rv (virulent) and H37Ra (partially attenuated ) strains of M. tuberculosis, and their DNA sequence was determined. A host–vector system suitable for the production of sufficient quantities of the recombinant AlaDH antigen was developed. The AlaDH gene was expressed under the control of strong, transcriptional (bacteriophage pLpR) and translational (atpE ) signals. High-level expression of soluble AlaDH was obtained using the recombinant E. coli K-12 strain CAG629 [pMSK12], which is deficient in Lon protease and the heat-shock response. A simple two-step procedure for the rapid purification of the recombinant protein was developed. The protein was purified to near homogeneity, and the purified AlaDH showed a specific enzyme activity comparable to the native protein isolated from M. tuberculosis. In addition, the product showed an expected amino acid sequence and reacted strongly to the 40-kDa (AlaDH )specific mAb HBT-10. Furthermore, the epitope of the mAb HBT-10 was mapped to a 12-amino-acid region. Contrary to the published results, we show that the AlaDH and the PNT (pyridine nucleotide transhydrogenase) of M. tuberculosis do not share common epitopes reacting to the species-specific mAb HBT-10. The availability of highly purified AlaDH should now enable a detailed biochemical and structural characterization of this important enzyme of M. tuberculosis. © 1998 Elsevier Science B.V. All rights reserved. Keywords: 40 kDa; PCR; Cloning; Epitope; HBT-10; Antigen

1. Introduction Mycobacterium tuberculosis, the causative agent of human tuberculosis, has re-emerged as a major world * Corresponding author. Tel: +49 531 6181 320; Fax: +49 531 6181 458/515; e-mail: [email protected] Abbreviations: AlaDH, alanine dehydrogenase; Ap, Ampicillin; BCIG, 5-bromo-4-chloro-3-indolyl--galactopyranoside; Boc, tert-butyloxycarbonyl; DICD, diisopropylcarbodiimid; DMF, dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; FPLC, fast protein liquid chromatography; Gly, glycine; HIV, human immuno deficiency virus; HOBt, hydroxybenzotriazol; IB, inclusion body; mAb, monoclonal antibody; MTT, thiazolyl blue tetrazolium bromide; NCA, N-carboxyanhydride; NMP, 1-methyl-3-pyrrolidinon; OtBu, tert-butylester; PAGE, polyacrylamide gelelectrophoresis; PBS, phosphate-buffered saline; Pfp, pentaflourophenyl; Pmc, pentamethylchroman; PNT, pyridine nucleotide transhydrogenase; rec, recombinant; SDS, sodiumdodecylsulafte; TB, tuberculosis; TBS, Tris-buffered saline; Trt, trityl. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 13 4 - 6

health problem (Bloom and Murray, 1992). Although TB is more prevalent in developing countries where adequate public health systems are not available (Grzybowski, 1991; Kochi, 1991), developed countries are also experiencing increasing TB, especially in immuno-compromised persons, e.g. HIV-infected individuals (Selwyn et al., 1989; Barnes et al., 1991). An additional problem has been the widespread appearance of multiple drug-resistant strains of M. tuberculosis. Better understanding of pathogenic mechanisms and availability of rapid diagnostic tests and more effective vaccines are some of the main goals of current research on TB. Species-specific and secretory antigens released early during culture and infection are of considerable interest for the purpose of vaccine development and diagnosis of TB (Andersen et al., 1991; Andersen, 1994). The 40-kDa antigen has been confirmed as -alanine dehy-

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B. Hutter, M. Singh / Gene 212 (1998) 21–29

drogenase ( EC 1.4.1.1; Andersen et al., 1992). It is a secreted protein of M. tuberculosis and is absent in the vaccine strain M. bovis BCG (Andersen et al., 1992). The mAb, HBT-10 (Ljungqvist et al., 1988), which is highly specific for the 40-kDa antigen has a unique feature of distinguishing M. tuberculosis from M. bovis BCG. In addition, AlaDH may be involved in the virulence of M. tuberculosis. As -alanine is a constituent of the mycobacterial peptidoglycan, it was suggested that AlaDH may play an important role in the synthesis of -alanine, which is the precursor of -alanine (Andersen et al., 1992). Interestingly, although M. tuberculosis is a mesophilic organism, its AlaDH is highly thermostable. We are interested in a detailed functional and structural characterization of AlaDH of M. tuberculosis. For this purpose, sufficient quantities of the purified protein are required. Purification of AlaDH from M. tuberculosis is very difficult due to the virulent nature and slow growth rate of the organism. In this study, we describe cloning, sequencing and heterologous expression of the gene coding for AlaDH of M. tuberculosis. Discrepancies in the DNA sequence published previously were detected. A rapid purification procedure for recAlaDH and data on epitope mapping of the species-specific mAb HBT-10 are also presented.

2. Materials and methods

the culture temperature to 42°C for 60–180 min in a water bath shaker. Bacteria were harvested by centrifugation at 6000×g. The bacterial pellet was resuspended in 20 mM Tris/HCl, pH 8.1, and cells were broken by sonication in ice (3×2.5 min for 1-l cultures) using a Braun Labsonic 2000. In order to eliminate intact bacteria and insoluble bacterial compounds, the suspension was centrifuged twice for 30 min at 47 000×g at 4°C. The supernatant was passed through a 0.2-mm membrane (Minisart, Sartorius) before it was used for further experiments. 2.3. PCR amplification The clone lAA65, which contains the AlaDH of M. tuberculosis, was subjected to PCR amplification with the primers Clon-SphI (5∞-CCCGCATGCGCGTCGGTATTCCGACC-3∞) and ClonNotI/SfiI (5∞GGCCNNNNNGGCCGCGGCCGCTCAGGCCAGCACGCTGGCGGGCTCGGTGAACGGCAC-3∞). A three-step PCR procedure consisting of 30 cycles was used with a melting step for 1 min at 96°C, an annealing step for 1 min at 53°C and an extension step for 3 min at 72°C. The MgCl concentration was 2.5 mM. 2 2.4. Minipreps and Maxipreps DNA extraction techniques were essentially the same as reported earlier (Sambrook et al., 1989; Singh et al., 1992).

2.1. Bacterial strains For expression experiments, the strains listed in Table 1 were used. 2.2. Media and growth conditions For the cultivation of all bacterial strains, LB medium containing Ap (100 mg/ml ) was used. Medium and additives were prepared by standard techniques (Sambrook et al., 1989). For the induction of recombinant ALaDH, cells were grown at 30°C to an A of 0.6, followed by shifting 600

2.5. DNA sequencing and Nucleotide Sequence Accession No. Sequencing was carried out using a 373A DNA Sequencer (Applied Biosystems). Reactions were prepared with the ABI PRISM@ Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer), following the manufacturer’s instructions. Computer analyses of the nucleotide and amino acid sequences were performed using DNASIS Software (Hitachi Software Engineering America, San Bruno, CA; Release 2.1). The nucleotide sequence has been deposited in the

Table 1 Bacterial strains used in this study are listed below Strain E. E. E. E.

coli coli coli coli

CAG 629 DH5a TG2 SURE

E. coli BL 321 E. coli N 4830 E. coli 538

Genotype/relevant phenotype

Source/reference

lac(am) pho(am) trp(am) supCts rpsL mal(am) lon htpR165-Tn10 supE44 DlacU169(w80 lacZ DM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 supE hsdD5 thi D( lac-proAB) D(srl-recA)306::Tn10(TetR) F ∞(traD36 proA+ lacI q lacZM15) hsdR mcrA mcrB mvr endA supE44 thi-1 l-gyrA96 relA1 lac recB recJ sbcC umuC uvrC [F ∞proAB lacI qZ DM15 Tn10(TetR)] rnc105 nadB+ purI + su° his ilv galKD8 DchlD-pgl (l DBam N+ cI DHI ) ts857 Genotype unknown

C. Gross, Wisconsin Hanahan (1983) Sambrook et al. (1989) Stratagene Studier (1975) Gottesman et al. (1980) Bayer AG

B. Hutter, M. Singh / Gene 212 (1998) 21–29

GenBank nucleotide sequence database under the Accession No. U92472. 2.6. Protein purification Debris-free soluble cell extract was purified on a FPLC-System (Pharmacia) using FPLC-Director software. The purification media used were Q-SepharoseHP (Pharmacia) and Blue Sepharose CL-6B (Pharmacia). Protein gels were prepared by standard procedures (Laemmli, 1970). Twelve-per-cent gels were used throughout the study. The gels were silver-stained using the Silver Stain Plus Kit (BioRad ). The protein concentration was determined using a Protein Assay Kit (BioRad ).

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2.9. Epitope mapping Oligopeptides were synthesized directly on a cellulose membrane (Spot-synthesis) as described previously ( Frank, 1992). Glycine and arginine were used as free HOBt-ester. Valine, leucine and isoleucine as NCA. All other amino acids were Fmoc-Pfp-ester. To protect the side chains of the amino acids, we used Pmc for arginine, Trt for asparagine, cysteine glutamine and histidine, OtBu for glutamate and aspartate and Boc for lysine. Treatment of the filter with mAb HBT-10 and detection using alkaline phosphatase coupled secondary antibody were performed according to Frank (1992).

2.7. Enzyme assay and activity staining in native gels The standard reaction mixture for oxidative deamination under saturating concentrations of substrates contained 125 mM glycine KOH, pH 10.2/100 mM -alanine/2.5 mM NAD+) and enzyme in a final volume of 1.0 ml (Ohshima et al., 1990). The reaction mixture was preincubated at 37°C before starting the reaction upon addition of protein followed by measuring the change in absorbance at 340 nm with a spectrophotometer. Protein bands possessing AlaDH activity were detected by activity staining of proteins separated on non-denaturing gels (7.5% polyacrylamide). All buffers and solutions were devoid of SDS and dithiothreitol. After electrophoresis, the gel was incubated at 37°C in developing solution (50 mM glycine KOH, pH 10.2/50 mM -alanine/0.625 mM NAD+/0.064 mM phenazine methosulfate/0.24 mM nitroblue tetrazolium) for 5 min (Andersen et al., 1992). A positive reaction was indicated by a purple colour on the gel. 2.8. Immunoblotting Proteins were separated on a 12% SDS–PAGE as described above. Then, they were transferred on to a membrane ( Immobilon@ PVDF, Millipore) with a semidry electrophoretic transfer cell (BioRad ) following the manufacturer’s instructions. Non-specific binding was blocked by incubating the membrane overnight in T-TBS (50 mM Tris HCl, pH 7.4/137 mM NaCl/3 mM KCl/0.05% Tween20), containing a 20% solution of milk (0.3% fat). Primary antibody, mAb HBT-10 ˚ .B. Andersen, Copenhagen), was diluted 1:1500 (from A in T-TBS and incubated for 1 h. After washing the filters three times, the secondary antibody, horseradish-peroxidase conjugated goat anti-mouse IgG (BioRad ), was diluted 1:3000 in T-TBS and incubated for 20 min. For detection, the ECL Western Blotting Detection Kit (Amersham) was used, following the manufacturer’s instructions.

3. Results and discussion 3.1. DNA sequence of the AlaDH gene Before constructing the expression clones, we decided to verify the published DNA sequence of the AlaDH gene M. tuberculosis. A complete DNA sequence of the gene present in the clone AA65 (Andersen et al., 1992) was obtained for both strands. We noticed differences between our sequence and that published by Andersen et al. (1992), e.g. at position 103 ( Fig. 1), the nucleotides AATTCC are absent. Thus, the AlaDH gene of M. tuberculosis consists of 1113 nucleotides coding for a protein of 371 aa. No putative signal sequence is present in AlaDH. We also verified the DNA sequence by amplifying the gene from the chromosome of M. tuberculosis H37Rv and the avirulent strain H37Ra. Furthermore, the DNA sequence of a PCR product obtained from the AA65 as well as the amino acid sequence of the corresponding recombinant protein confirmed that the sequence determined in this study ( Fig. 1) is unambiguous.

3.2. Construction of expression plasmid pMSK12 The DNA of clone AA65 (Andersen et al., 1992) was used as a template for amplifying the gene coding for recAlaDH by PCR. The primers used introduced cleavage sites for the restriction endonucleases SphI and NotI directly adjacent to the 5∞ and the 3∞ ends of the ORF, respectively (Fig. 2). The amplification product was digested with NdeI and SphI and then used for ligation to the vector. We constructed the vector pJLA604Not by the insertion of an adapter containing NotI site between the SphI and EcoRI sites of pJLA604 (Schauder et al., 1987). The amplification product was cloned between SphI and NotI sites of pJLA604Not ( Fig. 2).

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B. Hutter, M. Singh / Gene 212 (1998) 21–29

Fig. 1. Nucleotide sequence of AlaDH of M. tuberculosis (above) and the deduced aa sequence (below) is shown. Sequencing of both strands was conducted using an Applied Biosystems 373A sequencer. The DNASIS program package (Hitachi Software Corporation, USA) was used to analyze the sequence. The nucleotide sequence has been deposited in the GenBank nucleotide sequence database under the Accession No. U92472.

3.3. Expression of recAlaDH Seven different strains of E. coli were electroporated with the expression plasmid pMSK12. The production of the recombinant antigen was examined in each of these strains upon shifting the temperature from 30°C to 42°C. The results are summarized in Table 2. The

strongest expression was observed in the E. coli strain CAG629 (lon, htpR::Tn10). Extracts of this strain carrying the plasmid pMSK12 contained significant amounts of the recombinant protein after induction at 42°C as demonstrated by immunoblotting with mAb HBT-10 ( Fig. 3A, lane 2) and enzyme activity (Fig. 3B, lane 2). Most of the protein was present in the soluble cell

B. Hutter, M. Singh / Gene 212 (1998) 21–29

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Fig. 2. Construction of the expression plasmid pMSK12. The vector pJLA604Not is a derivative of pJLA604 (Schauder et al., 1987). pJLA604 was digested with EcoRI and SphI. An adapter containing, SphI NotI, SfiI and EcoRI sites was also cleaved with SphI and EcoRI followed by ligation with the vector using T4-DNA-Ligase (Boehringer Mannheim). The recombinant plasmid thus obtained was named pJLA604Not. The clone %AA65 was subjected for PCR amplification with the primers Clon–SphI (5∞-CCCGCATGCGCGTCGGTATTCCGACC-3∞) and Clon–NotI/SfiI (5∞GGCCNNNNNGGCCGCGGCCGCTCAGGCCAGCACGCTGGCGGGCTCGGTGAACGGCAC-3∞). The vector pJLA604Not and the PCR product were cleaved with SphI and NotI and then ligated, yielding the expression plasmid pMSK12.

fraction, although at least a part of the recAlaDH could also be detected in the cell pellet fraction (data not shown). High-level expression of other recombinant mycobacterial antigens has been reported earlier in this E. coli strain (Singh et al., 1992), but unlike previous reports, the recALaDH was produced mainly as soluble protein. Enzymatic tests in native gels showed a strong positive reaction for ALaDH, whereas uninduced cells were negative (Fig. 3B, lane 1). As a positive control, -alanine-dehydrogenase from Bacillus subtilis (Sigma)

was used. (Fig. 3B, lane 3). This enzyme showed no reactivity with the mAb HBT-10 (Fig. 3A, lane 3). For all further experiments, the recombinant strain CAG629 (pMSK12) was used. Unlike M. tuberculosis, the recAlaDH was not secreted in the medium by E. coli. 3.4. Purification of recALaDH Rapid purification of the recAlaDH from the E. coli soluble cell extract was achieved by a two-step pro-

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B. Hutter, M. Singh / Gene 212 (1998) 21–29

Table 2 Levels and type (soluble/insoluble) of biosynthesis of recAlaDH in different E. coli strains using the plasmid pMSK12 Strain E. E. E. E. E.

coli coli coli coli coli

Expression of recALADH CAG629 DH5a TG2 SURE BL321

E. coli 538 E. coli N4830

Highest expression, mostly soluble recAlaDH Low expression, mostly soluble recALADH Moderate expression, mostly soluble recALADH Low expression, mostly soluble recALADH Low expression, all the recALADH in IB’s (insoluble) Moderate expression, large part of recALADH as IB’s (insoluble) Moderate expression, about half of the recALADH is soluble

The term ‘low’ expression is used for an amount less than 3% of the total protein, ‘moderate’ expression refers to 3–5%, and a ‘high’ expression level for 5% and more as determined by densitometric tracing (Image Quant, Molecular Dynamics).

cedure. The first step was anion exchange chromatography on Q-SepharoseHP (Pharmacia). Using step gradients of NaCl, the protein was eluted between 250 and 300 mM KCl with a recovery of about 75% (Fig. 4). The second step consisted of affinity chromatography on Blue Sepharose CL-6B (Pharmacia). Since the buffer systems for both the ion exchange and the affinity columns were identical, it was sufficient to dilute the pooled active fractions from the ion-exchange column five times with loading buffer before application to a Blue Sepharose column. The protein was eluted using a linear gradient of NaCl ( Fig. 5). The column fractions showing enzyme activity were pooled. The thus purified recAlaDH was nearly homogenous as judged by silver staining of the protein in SDS–PAGE ( Fig. 6, lane 4). Results of the purification steps are presented in Table 3. Using the rapid purification procedure described above, about 32% recovery of active recAlaDH from E. coli was obtained. Thus, in shake cultures under laboratory

Fig. 3. Expression of the recAlaDH gene in E. coli CAG629 in a batch culture. (A) Western Blot using the mAb HBT-10. (B) Non-denaturing PAGE demonstrating -alanine dehydrogenase activity. Lane 1: non-induced cells of E. coli CAG629 (pMSK12). Lane 2: E.coli CAG629 (pMSK12) induced for 90 min at 42°C. Lane 3: -alanine dehydrogenase from Bacillus subtilis (Sigma) as control. Cells were grown in LB medium containing Ap (100 mg Ap/ml ) at 30°C to an A of 0.6. Induction was achieved by shifting the temperature to 42°C for 90 min in a shaking water bath. 600 Bacteria were harvested by centrifugation at 6000×g. The bacterial pellet was resuspended in 20 mM Tris/HCl, pH 8,1 and broken by sonication in ice (3×2.5 min for 1 l culture) using a sonicator (Braun Labsonic 2000). To get rid of intact bacteria and insoluble bacterial compounds, the suspension was centrifuged twice for 30 min at 47 000×g and 4°C. The supernatant was passed through a 0.2-mm membrane (Minisart, Sartorius) before it was used for further experiments. Ten microlitres of the sample were mixed 1:1 in sample buffer (100 mM Tris–HCl pH 6.8/200 mM dithiothreitol/4% SDS/0.002% Bromophenol Blue/20% glycerine) and heated at 95°C for 5 min before running them on 12% SDS–PAGE (Laemmli, 1970). Polypeptides were visualized by silver staining (BioRad Silver Stain Plus Kit). For immunoblotting, proteins were transferred on to a membrane (Immobilon@ PVDF, Millipore) with a semi-dry electrophoretic transfer cell (BioRad) following the manufacturer’s instructions. Nonspecific binding was blocked by incubating the membrane overnight in T-TBS (50 mM Tris–HCl, pH 7.4/137 mM NaCl/3 mM KCl/0.05% Tween20) containing a 20% solution of milk (0.3% fat). Primary antibody, mAb HBT-10 was diluted 1:1500 in T-TBS and incubated with the filter for 1 h. After washing the filter, horseradish-peroxidase conjugated goat anti-mouse IgG (BioRad), was added at a dilution of 1:3000 in T-TBS and incubated for 20 min. For detection, the ECL Western Blotting Detection Kit (Amersham) was used following the manufacturer’s instructions. To demonstrate the enzyme’s activity, non-denaturing PAGE (7.5% polyacrylamide) was used. All buffers and solutions were devoid of SDS and dithiothreitol. After electrophoresis, the gel was incubated at 37°C in developing solution (50 mM glycine KOH, pH 10.2/50 mM -alanine/0.625 mM NAD+/0.064 mM phenazine methosulfate/0.24 mM nitroblue tetrazolium) for 5 min. A positive reaction was indicated by the purple colour on the gel.

B. Hutter, M. Singh / Gene 212 (1998) 21–29

Fig. 4. Anion exchange chromatography of an E. coli CAG629 (pMSK12) soluble cell extract on Q-SepharoseHP (Pharmacia). The column was equilibrated with buffer A (20 mM Tris–HCl, pH 8.1) before the sample was applied to the column at a flow rate of 1 ml/min. An elution was performed using step gradients with buffer B (buffer A+1 M KCl ). After each step gradient, the column was washed until there was no more change of A . Each step gradient was collected as 280 a single fraction. The arrow indicates the fraction that contains -alanine-dehydrogenase activity.

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Fig. 6. Silver-stained SDS–PAGE gels of pooled fractions from different purification steps. Lane 1: molecular weight markers (Dalton Marker VII-L@, Sigma); Lane 2: crude extract. Lane 3: active fraction after anion exchange chromatography on Q-SepharoseHP. Lane 4: purified protein after subsequent chromatography on Blue Sepharose CL-6B.

KNNEFRVAITPA. This sequence is in complete agreement with the amino acid sequence of AlaDH derived from the DNA sequence of AlaDH gene determined in this study (Fig. 1). 3.5. HBT-10 does not cross-react to PNT

Fig. 5. Affinity chromatography of the active fractions from anion exchange chromatography (Fig. 4) on Blue Sepharose CL-6B (Pharmacia). The buffers used were the same as those for anion exchange chromatography. The sample was diluted five times with buffer A, and the conductivity was checked before the sample was applied to the column at a flow rate of 1 ml/min. The eluted material was collected as 1-ml fractions. Each fraction was analysed separately. The arrow indicates the peak that contains -alanine-dehydrogenase activity.

conditions, about 2 mg of active and homogenous recAlaDH could be obtained from 1 l of culture. N-terminal amino acid sequencing of the purified enzyme further confirmed the identity of the protein and yielded the following sequence: MRVGIPTET-

Delforge et al. (1993) reported an apparent crossreaction of mAb HBT-10 to pyridine nucleotide transhydrogenase (PNT; EC 1.6.1.1) in crude extracts of Mycobacterium tuberculosis. The molecular weight of the reacting band in Western blots (102 kDa) was in the range of PNT previously reported by Clarke et al. (1986) and Yamaguchi et al. (1988). Since HBT-10 is a highly specific antibody potentially useful for strain differentiation, we decided to investigate the cross-reaction reported by Delforge et al. (1993) in detail. We prepared a Western Blot using the culture filtrate from M. tuberculosis H37Rv and could also detect an approximately 100-kDa large band cross-reacting to mAb HBT-10. However, the N-terminal sequencing of this band revealed that the first 20 amino acids were completely identical to the N-terminus of the AlaDH (40-kDa antigen) of M. tuberculosis. In addition, this sequence was clearly different from the N-terminal sequence of PNT from M. tuberculosis recently characterized by Deshpande et al. (1994). This indicated that

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B. Hutter, M. Singh / Gene 212 (1998) 21–29

Table 3 Summary of purification steps and yields of the recAlaDH from E. coli Step

Total protein (mg)

Total activity ( U )

Specific activity ( U/mg)

Yield (%)

Purification factor

Crude extract Q-SepharoseHP Blue Sepharose CL-6B

163.8 12.5 1.1

80.7 60.4 26.0

0.49 4.84 23.72

100 74.8 32.2

1 9.8 47.5

Fig. 7. Mapping of epitope of mAb HBT-10. As described in the Materials and methods, 92 oligopeptides (12-mers), each overlapping with the neighbouring spot in eight amino acids, were synthesized directly on a cellulose membrane. The sequence of the 40-kDa antigen begins in the upper left corner and proceeds to the lower right corner. The mAb HBT-10 was used as primary antibody (diluted 1:33, incubation period 3.5 h). The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse IgG+IgM (H+L) (Jackson Immuno Research, diluted 1:500; incubation period: 1.5 h). Details of incubation conditions with the antibodies and the detection procedure were essentially according to Frank (1992). The spot a-12 that reacted strongly to mAb HBT-10 harbours the peptide stretch SAITDADFKAAG. This reaction was specific since no reaction to any of the oligopeptides was observed when HBT-10 was omitted.

the HBT-10 cross-reaction was in fact due to an AlaDH multimer, most likely a trimer and not to PNT. We further confirmed our finding by mapping the antigenic determinant of the mAb HBT-10. Using a solid phase method of synthesis, 92 oligopeptides (12-mers) were synthesized on to a cellulose membrane as defined spots. Each oligopeptide contained an overlap of eight amino acids with the preceding oligopeptide, thereby spanning the whole protein sequence of the AlaDH on 92 spots. Of the 92 spots, only one spot reacted with mAb HBT-10 (spot a-12, Fig. 7). The sequence of the corresponding oligopeptide is SAITDADFKAAG and represents amino acids 45–56 of the AlaDH of M. tuberculosis. This epitope was not detected in the amino acid sequence of PNT, further showing that HBT-10 would not crossreact to PNT of M. tuberculosis. 3.6. Conclusions

(1) The AlaDH gene of Mycobacterium tuberculosis was cloned and sequenced. Corrections in the previously published DNA sequence were made. We were able to obtain high level expression in E. coli CAG629

where the recAlaDH was produced as soluble and catalytically active protein. (2) A rapid purification procedure was established that resulted in nearly homogenous protein. The procedure is easy to perform, and the final yield of about 32% can be achieved. Thus, about 2 mg of active and homogenous recAlaDH could be obtained from 1 l of shake cultures of recombinant E. coli. (3) Contrary to the published results, we found that the mAb HBT-10 does not react to PNT of M. tuberculosis. This mAb is thus a highly specific antibody to AlaDH of M. tuberculosis. The epitope of HBT-10 was mapped on the AlaDH protein to a region of 12 aa (aa 45–56). This stretch of amino acids is not conserved in PNT. The HBT-10 epitope is distinct from the nucleotide binding site of the AlaDH.

Acknowledgement ¨ se Andersen, SSI, Copenhagen We are grateful to Dr A for providing us Lambda clones and the mAb HBT-10.

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