Purification of streptomycin adenylyltransferase from a recombinant Escherichia coli

Protein Expression and PuriWcation 40 (2005) 86–90 www.elsevier.com/locate/yprep

PuriWcation of streptomycin adenylyltransferase from a recombinant Escherichia coli Snehasis Jana, Goutam Karan, J.K. Deb¤ Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India Received 25 August 2004, and in revised form 4 October 2004 Available online 7 January 2005

Abstract Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat, contributing to interest in understanding the mechanisms of resistance. One important mechanism of streptomycin modiWcation is through ATP dependent O-adenylation, catalyzed by streptomycin adenylyltransferase (SMATase). The aim of this study was to purify the recombinant SMATase by Ni2+–IDA–His bind resin column chromatography. Thioredoxin-His6-tagged SMATase fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The puriWed fusion protein showed a single band on SDS–PAGE corresponding to 49 kDa. The recovery of fusion protein was 47% with ninefold puriWcation. The fusion system provided a single step, easy and very rapid puriWcation of SMATase and is suitable for obtaining a highly puriWed functional protein of interest. The fusion does not aVect the functionality of the protein.  2004 Elsevier Inc. All rights reserved. Keywords: AYnity chromatography; Trx-His6-tagged fusion system; Streptomycin adenylyltransferase

The aminoglycosides (Ags) are a family of molecules containing a molecular nucleus, an aminocyclitol ring that can be streptidine or 2-deoxystreptamine and two or more aminosugars linked by glycosidic bonds to the nucleus. The Wrst aminoglycoside, streptomycin, was discovered by Waksman, Schatz, and Bugie in 1944 and was isolated from Streptomyces griseous [1]. Many of the aminoglycosides used clinically are naturally occurring substances produced by actinomycetes of either the genus Streptomyces or Micromonospora [2]. Aminoglycosides are active primarily against aerobic gram-negative bacilli as well as gram-positive cocci. However, it is well known that bacteria develop resistance to antibiotics and multiresistant isolates have seriously increased [3]. Their broad anti-microbial spectrum, rapid bactericidal action, and ability to act synergisti*

Corresponding author. Fax: +91 011 26582282. E-mail address: [email protected] (J.K. Deb).

1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.10.005

cally with other drugs have made them especially useful in the treatment of serious nosocomial infections. However, as with other drugs, their overuse and misuse led to the development of resistance in important microbial pathogens [4]. Although detailed studies on several aminoglycosidemodifying enzymes have been limited, some mechanistic and mutational studies have been carried out and the crystal structures of two acetyl transferases, a nucleotidyl transferase and a phosphotransferase have been reported [5]. Genes encoding aminoglycoside-modifying enzymes are often located in plasmids, which permits cell-to-cell dissemination of the aminoglycoside resistance trait. Furthermore, several of these genes are also included in transposons and integrons [6], which result in the rapid dissemination at molecular level. Structural studies of the aminoglycoside-modifying enzymes are limited to interpretations of primary sequence information. The crystal structure of aminogly-

S. Jana et al. / Protein Expression and PuriWcation 40 (2005) 86–90

coside phosphotransferase (3⬘), aminoglycoside acetyltransferase (6⬘), and aminoglycoside nucleotidy ltransferase (4⬘) are known [7]. However, the exact biochemical functions of the conserved motifs in these enzymes have yet to be conWrmed by more detailed structural studies. So far, there is no report of structural study of streptomycin adenylyltransferase (SMATase). Little is known about binding or catalysis in these enzymes. The molecular architecture of SMATase is of special interest since there is no information on 3D structure for this enzyme. Furthermore, the elucidation of the active site of SMATase can only provide insight into the mechanism of nucleotide phosphate transfer and may provide a template for drug design. To get all information regarding this enzyme we puriWed the enzyme. The goal of this study was to produce puriWed SMATase protein. We report here for the Wrst time that the active form of SMATase can be successfully puriWed in homogeneous form as fusion protein by a metal aYnity chromatography. The highly soluble, thioredoxin-His6-tagged fused form of SMATase provides a convenient tool for further studies.

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Production of soluble thioredoxin-His-SMATase fusion protein Transformed E. coli BL21 (DE3)/pETSm6 [8,9] cells were grown at 37 °C in Luria–Bertani medium with 100 g/ml ampicillin and 30 g/ml streptomycin to a density of A600 D 0.6–0.8 and with continuous shaking at 250 rpm. The expression of SMATase protein was induced with 0.5 mM isopropyl-1-thio--D-galactoside (IPTG; Bangalore Genei., India), and the induced cells were grown for an additional 3 h. The cells were harvested and were collected by centrifugation for 15 min at 10,000 rpm and stored at ¡20 °C. PuriWcation of recombinant protein

The Escherichia coli BL21 (DE3) strain (Novagen, USA) was used for expression of His6-tagged SMATase. Plasmid pET32Xa/Lic (Novagen) was used for construction of the expression system. Recombinant plasmid pETSm6 was constructed in our laboratory and used for this study. Ni2+–IDA–His bind resin column was purchased from Novagen, USA. The streptomycin sulfate was purchased from Sigma (USA) and Ampicillin was purchased from Ranbaxy (India). IPTG was procured from Bangalore Genie, Bangalore, India. [
-32P]ATP from BRAC, Mumbai, India. All media components were purchased from Hi Media Laboratories, Mumbai, India. All other chemicals were from E. Merck, Mumbai, India.

Bacterial cells from 1-L culture were resuspended in 40 ml binding buVer [20 mM Tris–HCl (pH 7.9), 500 mM NaCl, and 5 mM imidazole] containing a protease inhibitor PMSF (1 mM). The resulting suspension was treated with DNase I (2.5 mg/ml) and RNase A (2.5 mg/ml) for 20 min at room temperature. The suspension was subjected to disruption by sonication [10 bursts (18 kHz), each burst (15 m) for 30 s was alternated by a 30 s of intermittent cooling on ice. Cell debris was removed by centrifugation at 15,000 rpm for 30 min. PuriWcation was carried out by immobilized Ni2+-aYnity chromatography [10–13] as described in the supplier’s instructions. The resultant supernatant (extract) was passed through a 0.45 m syringe-end Wlter prior to loading. The lysate was loaded onto a column containing 1.25 ml of His-Bind Resin preequilibrated with binding buVer. Next, the column was washed extensively with washing buVer [20 mM Tris–HCl (pH 7.0), 500 mM NaCl, and 60 mM imidazole] to remove unbound and non-speciWc bound proteins. The protein was eluted with step concentration gradient of 80, 100, 250, 500 mM, and 1 M imidazole in the elution buVer [20 mM Tris–HCl (pH 7.9), 500 mM NaCl, and 80 mM– 1 M imidazole]. One-milliliter fractions was collected. All steps were carried out at 4 °C.

Construction of expression plasmid

SDS–polyacrylamide gel electrophoresis

The streptomycin resistance gene was obtained by cloning of partial Sau3A1 digested genomic DNA of a spontaneous streptomycin resistant mutant of Corynebacterium acetoacidophilum at the BamHI site of pTZ18U. The gene of interest was further trimmed by deletion using Exonuclease III, S1 nuclease (NEB, USA) and deletion mutants were subcloned in the expression vector pET32Xa/Lic, which was digested with NcoI and blunt ended by T4 DNA polymerase (NEB, USA). Streptomycin resistance recombinants were selected. The smallest of these clones (pETSm6) was used for expression study.

The fractions obtained in diVerent puriWcation steps were analyzed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). SDS–PAGE (12.0% gel) was performed with the buVer system of Laemmli [17]. The proteins were stained with Coomassie brilliant blue R-250.

Materials and method Bacterial strains, plasmids, and reagents

Protein concentration determination Protein concentration was determined by the Lowry method [18]. Bovine serum albumin was used as a standard.

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Western blot Following electrophoresis of the puriWed protein sample, it was transferred to a nitrocellulose membrane for immunostaining [19]. The membrane was blocked with PBS containing 0.1% Tween 20 (PBST) and 3% BSA (blocking buVer) for 2 h, washed with PBST, and then incubated with the primary antibodies [monoclonal antihis antibody (Clontech Laboratories) diluted 1:2000], which was diluted in blocking buVer for 2 h. After extensive washing with PBST, the membranes were incubated for 1 h with the secondary antibodies, horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Biotech) diluted 1:10,000 in blocking buVer and thereafter washed in PBST and water. All steps were carried out at room temperature. Antibody bound protein was detected with chemiluminescent substrate (SuperSignal, Pierce). Enzymatic assay The enzymatic activities of the cell extract and puriWed samples were determined by the method of Hass and Dowding [20]. The assay mixture contained 10 l assay buVer [20 mM Tris–Cl, pH 7.8, 1.25 mM MgCl2, 0.5 mM NH4Cl, and 1 mM EDTA], 10 l [
-32P]ATP mixture, 10 l enzyme preparation, and 2 l streptomycin (1 mg/ml). The reaction mixtures were incubated at 37 °C for 20 min, and aliquot samples were withdrawn at various times. The samples withdrawn from assay mixtures were chilled and pipetted onto a membrane (1 cm2) of Whatman P-81 phosphocellulose paper. After 5 min at room temperature, papers were placed in 100 ml hot distilled water (70–80 °C) for 5 min. The liquid was then poured oV and repeated three times. The papers were rinsed four times with 100 ml distilled water and dried at room temperature. These were then placed in a 8 ml scintillation vials containing 4 ml scintillation cocktail-O (contents per liter: 6 g PPO and 0.2 g POPOP in toluene) and counted by Beckman LS 9800 liquid scintillation counter (Beckman).

Fig. 1. SDS–PAGE gel (12%, Coomassie stained) of crude E. coli cell extracts expressing thioredoxin-His6-tagged SMATase fusion protein of pETSm6. Lane 1, uninduced cells (pET32); lane 2, induced cells (pET32); lane 3, uninduced pETSm6; lane 4, induced pETSm6 (0.5 mM IPTG); after 3 h of induction at 37 °C; and lane 5, molecular weight markers.

Results and discussion The presence of fusion protein in IPTG-induced E. coli BL21 (DE3) strain bearing plasmid pETSm, which was derived from a clone of SMATase gene isolated from spontaneous streptomycin resistant mutant of C. acetoacidophilum (data not shown) was studied by SDS– PAGE. Trx-His6-tagged protein (19 kDa, [9]) is visible in the total protein sample of the cells containing the parent plasmid, pET32 (Fig. 1, lane 2). A 49 kDa band (Trx-His6-tagged SMATase protein) was observed in SDS–PAGE of total protein sample (Fig. 1, lane 4) of the cells containing pETSm6. The size of the observed

Fig. 2. SDS–PAGE analysis of the puriWed Trx-His-tag-SMATase. Gel stained with Coomassie blue R-250. Lane 1, soluble fraction; lane 2, Xowthrough fraction; lane 3, wash fraction; lane 4, elute fraction (500 mM imidazole) from Ni2+ column; and lane 5, molecular weight marker.

fusion protein is in accordance with the size of the fusion protein (calculated from the amino acid sequence) comprised Trx-His (19 kDa) and SMATase (30 kDa).

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Table 1 PuriWcation of Trx-His-tagged-SMATase protein from 1-L culture of E. coli. Fraction

Volume (ml)

Total protein (mg)

Total activity (Ua)

SpeciWc activity (U/mg)

Recovery or yieldb (%)

PuriWcation foldc

Cell lysate Ni2+ column

40 10

53 1.3

31,902 14,974

602 11,518

100 47

1 19

a

One unit of enzyme is deWned as the amount of enzyme necessary to produce 1 pmol AMP-streptomycin per minute per milligram of enzyme at 35 °C in standard assay condition. b Recovery or yield D (total activity of elute fraction/total activity of cell lysate). c PuriWcation factor D (speciWc activity of elute fraction/speciWc activity of cell lysate).

The recombinant protein was puriWed with native Ni2+–IDA–His bind resin column chromatography [14– 16]. The use of pET32 vector and the aYnity chromatography of the resulting fusion protein permitted rapid and easy production of Trx-His-SMATase. Pure protein was obtained in fractions eluted with 500 mM imidazole during elution. The puriWed protein appeared as a single band on 12% SDS–PAGE (Fig. 2), indicating that it is electrophoretically pure (99%). The total protein concentration and the puriWed fusion protein samples was determined by the Lowry method using BSA as a standard. About 1.3 mg of pure His6-tagged SMATase from 1-L culture E. coli BL21 (DE3) was obtained by this protocol. The results of puriWcation of the recombinant fusion protein are summarized in Table 1. To identify the expressed protein, we used anti-His antibody that recognizes continuous six histidines repeat. The Western blot analysis indicated that the recombinant protein is a positive His-tagged fusion protein (Fig. 3). Trx-His6-tagged SMATase was puriWed by over 19-fold with a speciWc activity of about

Fig. 4. Streptomycin adenylyltransferase activities in crude extract and puriWed fraction of E. coli BL21 (DE3) and recombinant E. coli BL21 (DE3)/pETSm6.

11,518 pmol/min/mg protein. The recovery of fusion protein was 47%. The enzyme activities of the extracts prepared from non-recombinant E. coli (pET32), recombinant E. coli (pETSm6), and puriWed protein are shown in Fig. 4. Although total CPM in puriWed sample was less than that of crude (pETSm6) extract, the speciWc activity of puriWed sample was higher than crude sample (Table 1). These results clearly indicated that the adenylation of streptomycin was due to the presence of SMATase enzyme and conWrmed that the presence on the N-terminus of 171 additional amino acids (fusion part) has no inXuence on the SMATase activity (Fig. 4). Fig. 3. Western blot analysis of histidine-tagged fusion protein of the SMATase. PuriWed SMATase from SDS–PAGE after electrophoresis using a 12% polyacrylamide gel was transferred to Immun-Blot nitrocellulose membrane. The membrane was incubated in 3% BSA and then incubated in 1:2000 dilution of mouse anti-His antibody in the blocking buVer. After washing, the membrane was incubated in 1:10,000 dilution of horseradish peroxidase-conjugated sheep antimouse IgG in the blocking buVer. Antibody bound protein was detected with chemiluminescent substrate (SuperSignal, Pierce). (A) PuriWed fusion protein (lane 1) and molecular weight marker (lane 2) in 12% SDS–PAGE. (B) Western blot of puriWed histidine-tagged fusion protein of the SMATase.

Conclusions For the Wrst time, a single-step puriWcation protocol for active SMATase has been accomplished. Application of only IMAC chromatography on Ni2+–IDA–His bind resin for puriWcation of His6-tagged SMATase protein is suYcient for obtaining pure protein that exhibits 99% purity. The presence of the N-terminal fusion domain

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has no eVect on SMATase activity. The puriWed recombinant SMATase fusion protein could be used for future structural studies.

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