Manzur, M. Electronic Journal et al. of Biotechnology ISSN: 0717-3458 Special 2006 Universidad Católica de Valparaíso -- Chile © 2006 Issue, by Pontificia Abbreviations: Ang II: AngiotensinRESEARCH II DOI: 10.2225/vol9-issue3-16 AT : Angiotensin II type receptor GAPDH: Glyseraldehyde-3-phosphate dehydrogenase Vol.9 No.3, ARTICLE MMLV: Moloney murine Leukemia Virus PND: post-natal day SN: supernatant Production of recombinant enzymes of wide use for research For biotechnological purposes, protein expression refers to María manipulate J. Manzur DNA in defined ways (Thatcher and Departamento de Bioquímica y Ciencias Biológicas to the directed synthesis of large amounts ofHitchcock, desired 1994). The major tools for genetic engineering Facultad de Química, Bioquímica y Farmacia proteins. The aim of the present work was to produce are the enzymes that catalyze specific reactions on Universidad Nacional de San Luis reverse transcriptase Moloney murine Leukaemia Virus DNA/RNA DNA molecules. polymerase Taq and Moloney Ejército de los Andes 950 retro-transcriptase and TaqDNA polymerase, murine asLeukaemia Virus (MMLV) retrotranscriptase are San Luis, Argentina 54 2652 422644 bioactive products. In the present paper, wewidely reportTel/Fax: used the enzymes for research in laboratories applying E-mail: mjmanzu@unsl.edu.ar preparation of recombinant enzymes, expressed molecular in E. biology methods. Recombinant enzymes are colistrains. The enzymes produced exhibited quite available goodin the market but at high prices. To reduce the Rosana V. Muñoz activity, compared with commercial enzymes, cost allowing of lab experiences, we made the effort to produce our de Bioquímica y Ciencias Biológicas us to replace the last ones for several labDepartamento applications. own recombinant enzymes. Facultad de Química, Bioquímica y Farmacia We a re reporting changes and modifications toUniversidad Nacional de San Luis de losof Andes 950 standard protocols described. The standard protocols TheEjército success modern biotechnology results from the San Luis, Argentina were modified, i.e.for the purification step ofability Taq, to express a foreign or heterologous genes in a host Tel/Fax: 54 2652 422644 temperature dependent procedure was designed.organism. The However, transcription and translation of a E-mail: munoz@bio.puc.cl enzymes produced were used in different applications, recombinant gene not always lead to the accumulation such as PCR, RT-PCR, PCR Multiplex andofRAPDs a folded fully active protein (Price and Stevens, 1999). It Adrián A. Lucero molecular markers. is well-known that artificially indu ced abnormal proteins, Departamento de Bioquímica y Ciencias Biológicas Facultad de Química, Bioquímica y Farmacia as well as foreign proteins accumulate in an insoluble state, Universidad Nacional de San Luis known as inclusion bodies, which contain almost pure Ejército de los Andes 950 Protein expression refers to the directed synthesisprotein of large held together by non covalent force which could San Luis, Argentina amounts of desired proteins. Many of the revolutionary only be solubilized strong denaturing agents (Thatcher Tel/Fax: 54 2652with 422644 E-mail: adrian_lucerhoff@yahoo.com changes that have occurred in the biological sciences and Hitchcock, over 1994). The biotechnology challenge is to th e past 15-20 years can be directly attrib utedexploit to the the ability inclusion body phenomenon, and to convert the Maximiliano Juri Ayub Departamento de Bioquímica y Ciencias Biológicas Facultad de Química, Bioquímica y Farmacia Universidad Nacional de San Luis Ejército de los Andes 950 San Luis, Argentina Tel/Fax: 54 2652 422644 E-mail: juriayub@dna.uba.ar Sergio E. Alvarez Departamento de Bioquímica y Ciencias Biológicas Facultad de Química, Bioquímica y Farmacia Universidad Nacional de San Luis Ejército de los Andes 950 San Luis, Argentina Tel/Fax: 54 2652 422644 E-mail: sealvar@un sl.edu.ar Gladys M. Ciuffo* Departamento de Bioquímica y Ciencias Biológicas Facultad de Química, Bioquímica y Farmacia Universidad Nacional de San Luis Ejército de los Andes 950 San Luis, Argentina Tel/Fax: 54 2652 422644 E-mail: gciuffo@unsl.edu.ar (a) SDS-PAGE (12.5%) of aliquots of the preparation at different purification steps. Lane 1: solubilized proteins after 11 *commercial 292 Keywords: Financial Figure hrs induction. (b) (c) 10). Corresponding Amplification SDS-PAGE ofLanes IPTG 1. Purification support: Lane 1-8: (1bioactivity, mM) volumes (12.5%, 2: Taq author products SN. obtained MW: of Grant silver of protein ofmolecular the from staining) after expression, AT Taq the Taq theweight Universidad of polimerase sonication employed thepurification, commercial ladder, step. Nacional and (µl): 1IIThis kb. receptor, Lane activity 1recombinant (lane (0.3), paper de 3: 1) proteins San 2assay. obtained and (0.4), isLuis, available enzymes. produced remaining Argentina. with (0.5),the on (lane (0.6), line produced after 2)at5purification http://www.ejbiotechnology.info/content/vol9/issue3/full/16/ (0.7), (0.8), by polimerase heat. (0.9 Taq (lanes Taq 1-8) (1). and polimerase. Lanes commercial 9-10: ), 0.3 one and (lanes 0.4 µl, 92 Ang Production of recombinant enzymes of wide use for research protein encapsulated into a useful bioactive product. It antibiotics, has spread on a plate and incubated at 37ºC. been suggested that protein deposited in these inclusions are aggregates of misfolded protein (Bowden et al. 1991; Expression induction with IPTG Chaffo tte et al. 1992; Thatcher and Hitchcock, 1994). Induction was performed for different times with Isopropil The aim of the present work was to produce reverse ß-thiogalactoside (IPTG, mM) in the appropriate culture transcriptase MMLV and TaqDNA polimerase, media. Expression as was controlled by analyzing aliquots of bioactive products. Thus, we set up a protocol for the material obtained at the different steps by SDS-PAGE expression of recombinant proteins in E. coli to obtain (12%). Once the best conditions for time, IPTG enzymes of high purity and specific activity. concentration We are and other variables were set up, a larger scale reporting changes and modifications to standard protocols culture was performed, which was used for protein described in the literature (Engelke et al. 1990; Pluthero, purification (Lawyer et al. 1989; Bollag et al. 1996 ; 1993; Ottino, 1998; Taube,1998). Ausubel et al. 1999). MATERIALS AND METHODS Purification Standard protocols were used for the production of Purification of Taqpolymerase. To purify Taq recombinant proteins including the following steps.polymerase we took advantage of the resistance of the enzyme to high temperatures and designed a purification Transformation of competent cells based on ofheating. bacteria The was resuspended pellet in PBS with mg/ml of lysozyme and the mixture was Competent cells were generated starting from exposed the strainto several E. cycles of frozen/melting steps to favour coliDH5a and BL21(DE3) by using the CaCl cellular standard breakage. After sonication (3 pulses), cellular protocol (Ausubel et al. 1999). Competent cells were lysates were centrifuged and the supernatant (SN) transformed using the vector pTTQ18 containingrecovered. the The SN was heated at 72ºC for hr and then sequence of Taqwith a selection marker for Ampiciline centrifuged polymerase at 15000 x remains g, Taq in the (Amp) and a vector containing the MMLV sequence SN. Purified and proteins were dialyzed against storage buffer selection markers for Chloranfenicol and Kanamycine (50 mM Tris-HCl (both pH = 8, 100 mM NaCl, 0.1 mM EDTA y vectors were genero usly provided by Ing. Masuelli, mM ß-mercaptoeth Fac. anol), in two steps, lasting three days. Cs. Agrarias, Mza). Transformation was carried Sterile outglycerol by was added to the dialyzed material to a final thermic shock: competent bacteria were incubated concentration with the of 50% to cryoprotect the enzyme and stored vector 10 on ice, followed by incubation at at 42ºC -20ºC. for 2Reaction buffer (10 x) free of Mg was prepared and a final step at 4ºC. The transformants were (10 mM Tris-HCl (pH 9.0), 50 mM KCl and 0.1%, Triton resuspended in 500 µl of culture media containing X-100). (b) Purification from inclusion bodies and dialysis with different triton X-100 concentrations. SDS-PAGE gels (7.5%), Figure (a) stained Induced with 2. Purification over CBB. expression of theofretrotranscriptase. MMLV in SN and inclusion bodies SDS-PAGE gels (7.5%), stained with CBB. 293 Manzur, M. et al. MMLV purification. Bacterial slurry was centrifuged at RESULTS AND DISCUSSION 4000 rpm for 10 and the pellet was resuspended in 30 ml wash buffer (50 mM Na HPO4 pH 8, 0.3 M NaCl, Following mMthe procedures described under Methods, the 2-mercaptoethanol). Cellular lysis was achieved recombinant by enzymes were expressed and purified from E. treatment with lysozyme mg/ml and sonication as DH5a and BL21 coli cultures (Figure and Figure 2). described above. Aliquots were centrifuged at 5000 Figure x shows gfor the purification steps followed to produce 15 min. SDS-PAGE analysis indicates that the protein polymerase of enzyme Taq(SDS-PAGE, Coomasie staining). in terest was present in the soluble fraction as well Figure as in 1bthe shows the silver staining of the commercial and inclusion body fractio n. Inclusion bodies were resuspended polymerasethe obtained Taq in this work. In order to test in wash buffer containing 0%, 0,5% and 2% of Tritonthe X-enzymatic activity of the enzyme we performed 100. Three washes with Triton X-100, followed by amplification receptor of thewith AT a commercial washes without detergent were performed. The pelletwas enzyme and compare with the amplification of AT2 resuspended in ml of solubilization buffer (50 mMreceptor Tris with increasing amounts (0.3 to µl) of the pH 8, M Urea, 0.3 M NaCl, mM 2-ß-mercaptoethanol). produced enzyme, following a previously described PCR Following centrifugation (12000 x g, hr)protocol the SN was (Ciuffo et al. 1996). Figure 1c shows amplification diluted in solubilization buffer and protein was renatured by products receptor (586 for the bp) AT with all the enzyme dialysis at 4ºC against 50-100 V of renaturation volumes buffer.used, The having a more specific amplification product dialyzed material was centrifuged at 13000 x g(1 with hr)the andprepared enzyme. A well-defined band of the the SN resuspended with the same volume of glycerol expected andsize was obtained with our enzyme. The signal stored at -20ºC. obtained with 0.4 µl of the enzyme was comparable to the one obtained with 0.3 µl of the commercial enzyme. From Activity assays these experiences, the estimated specific activity was 2-5 U/µl. In order to determin e the best assay conditions, The enzymatic activity was verified by means of different variable concentrations were included of MgClin the RT or PCR assays, using variable conditions: enzyme reaction mixture (data not shown). volume, MgCl concentrations, etc. Figure shows the over-expression of MMLV (65 kDa) PCR. Aliquots of DNA fro m adult rat kidney either wereinused the soluble to fraction (SN) or in the in clusion bodies amplify the AT receptor subtype of Ang II, following (pellet), with a higher yield in the inclusion bodies (Figure standard protocols to amplify the fragment of interest 2a). From the soluble material the enzyme was purified by (Dieffenbach and Dveksler, 1995; Nickenig et using al. 19 His-tag 97). affinity chromatography. However, a higher yield was obtained by purification starting from the RT-PCR assay. RNAs obtained from cerebellum inclusion of bodies. While most of the authors purify the different ages (TRIzol, GIBCO) were used toenzyme producefrom the soluble material (Sun et al. 1998; Taube et cDNA by retrotranscription in a first step (RT) and al. 1then 998), we decided to pursue the purification from the amplification was conducted for AT and GAPDH inclusion bodies. In Figure 2b it can be observed that a fragments by PCR assays as described (Ciuffo etconcentration al. 1996). of Triton X-100 0% to 0.5% gives a better yield on the purification process than a 2% of Triton X-100. RFLP. Amplification p roducts were digested with the indicated enzymes. Recombinant enzymes obtained in the lab were used to perform different amplification assays by using DNA from variable sources, such as animal (Figure 1c), vegetal or viral origin with excellent results (Pungitore et al. 2004). Figure shows an example where we analyzed the expression of two different genes by RT-PCR in a single assay (Multiplex PCR): simultaneous amplification was performed for the recep Angtor II (586 AT bp) 2and GAPDH (350 bp) genes, the second used as control. Both steps, the RT and the PCR were performed with the enzymes produced in the lab. These assays allow us to confirm that both enzymes are functional, since coDifferent previous amplification maximum development resultsof obtained expression the in two 2001) the stages by expression (Figure target at au were receptor PND15, toradiography sequences 3). analyzed level was in agreement ofobserved was and AT (Arce achieved. a change with 294 al. Figurepost-natal Co-amplification cerebellum (PND: Etidium independent 3.bromide RT-PCR atexperiences. different day) staining. of co-amplification ATand developmental C+: Experiment PND4. positive receptor Lower by stages. control. representative (586 PCR Panel: Upper bp) Multiplex. PND8 andPanel: of GAPDH tofour PND60. PND0 (350 bp) in et Production of recombinant enzymes of wide use for research The identity of the AT receptor fragmentspecific (586 bp)activity as shown by different assays performed. amplified from rat kidney DNA with our enzyme, was verified performing a restriction fragment length ACKNOWLEDGMENTS polymorphism (RFLP). Figure shows the digestion products of the 586 bp fragment with two differentM. Juri Ayub and S.E. Alvarez, have fellowships from enzymes. Fragments of the expected size were obtained,CONICET (Consejo Nacional de Investigaciones th us indicating the co rrect identity of the amplified Científicas y Técnicas, Arg). We thank to Dr. R. Masuelli fragment of AT receptor. for helpful suggestions. G.M. Ciuffo is a member of the CONICET researcher career. When th e goal is to express proteins as a reagent in biochemical or cell biology experiments, the authenticity of REFERENCES the protein function, such as high specific enzymatic activity is very important. The presen t resultsARCE, show that theSANCHEZ, S.; SELTZER, A. and CIUFFO, M.E.; enzymes obtained had their specific activity proved G.M. in Autoradiographic localization of angiotensin II different system and complex reactions such as the receptors in developing rat cerebellum and brainstem. Multiplex RT-PCR. Regulatory , 2001, Peptides vol. 99, no. 1, p. 53-60. Taqpolymerase was a soluble protein, a fact that simplifies Frederick M.; BRENT, Roger.; KINGSTON, AUSUBEL, the purification protocol. 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Inclusion bodies of the thermophillic endoglucanase D from Clostridium arethermocelliu made of native m enzyme that resistEuropean 8M urea.Journal of Biochemistry , 1992, vol. 205, p. 369-373. Figure 4. RFLP of the AT amplified fragment. fragment digested with PvuII, . Lanel 2: digestion with control product. MW molecular weight marker (100 bp ladder). CIUFFO, G.M.; JOHREN, O., EGIDY, G.; HEEMSKERK, F.M.J. and SAAVEDRA, J.M. Heterogeneity of rat Ang II Lane 1: AT receptors.AT In: RAIZADA, M.; PHILLIP, I. and SspI . C: SUMMERS, Recent Advances C. eds. in Angiotensin Receptors . Plenum Press, 1996, p. 189-197. Different approaches were used to purify MMLV DIEFFENBACH, C.W. and DVEKSLER, PCR G.S. retrotranscriptase, however, in this paper the best results Primer. A laboratory Cold Spring manual.Harbour: Laboratory Press, 1995. ISBN 0-87969-447-5. were obtained from the inclusion body fraction, while most of the authors use the soluble fraction. 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C+: positive control. 294 Production of recombinant enzymes of wide use for research The identity of the AT receptor fragment. protocols were used for the production of Purification of Taqpolymerase.To purifyTaq recombinant proteins including the following steps.polymerase we took advantage of the resistance of the enzyme. present paper, we report thewidely used enzymes for research in laboratories applying preparation of recombinant enzymes, expressed in E.molecular biology methods. Recombinant enzymes are colistrains.