Extremely Thermostable Serine-type Protease fromAquifex pyrophilus

Abstract

A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 °C, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent. A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 °C, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent. Hyperthermophiles are organisms that grow optimally at 80 °C or higher temperatures. Ever since they were identified at various geothermal locations, there has been interest in their evolutionary significance as well as their ability to thrive at temperatures in which normal proteins would be denatured. From the ability to grow autotrophically at high temperature and their position in the deepest branch of the phylogenetic tree, hyperthermophiles are presumed to be the closest living descendants of a universal ancestor of life (1Olson G.J. Woese C.R. Overbeek R. J. Bacteriol. 1994; 176: 1-6Crossref PubMed Google Scholar, 2Woese C.R. Kandler O. Wheelis M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4576-4579Crossref PubMed Scopus (4465) Google Scholar). Except for two genera of eubacteria, Aquifex andThermotoga, all identified hyperthermophiles belong to Archaea. Most of them are grouped as autotrophs that use inorganic materials such as carbon and nitrogen sources or heterotrophs that exploit organic materials as nutrients (3Stetter K.O. FEMS Microbiol. Rev. 1996; 18: 149-158Crossref Google Scholar). To degrade exogenous organic compounds, heterotrophic hyperthermophiles have various hydrolytic enzymes such as proteases and glycosidases. Autotrophic organisms also contain proteases that may be involved in cellular processes. The proteases from hyperthermophiles have been studied for their biotechnological applications as well as for their metabolic significance (4Bauer M.W. Halio S.B. Kelly R.M. Adv. Protein Chem. 1996; 48: 271-309Crossref PubMed Google Scholar). Among the existing types of proteases such as serine-, thiol-, or acid-type proteases, the majority of them belong to a family of serine-type proteases (5Cowan D.A. Smolenski K.A. Daniel R.M. Morgan H.W. Biochem. J. 1987; 247: 121-133Crossref PubMed Scopus (92) Google Scholar, 6Eggen R. Geerling R.A. Watts J. de Vos W.M. FEMS Microbiol. Lett. 1990; 71: 17-20Crossref Google Scholar, 7Mayr J. Lupas A. Kellermann J. Eckerskorn C. Baumeister W. Peters J. Curr. Biol. 1996; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Volkl P. Markiewicz P. Stetter K.O. Miller J.H. Protein Sci. 1994; 3: 1329-1340Crossref PubMed Scopus (55) Google Scholar, 9Voorhorst W.G.B. Warner A. de Vos W.M. Siezen R.J. Protein Eng. 1997; 10: 905-914Crossref PubMed Google Scholar). Some of them are processed from a preprotein to a mature form and are localized at the cell wall or are secreted. These proteases show heat stability or are resistant to a high concentration of denaturing agents. The half-life measured at around 100 °C varied from a few minutes for archaelysin (5Cowan D.A. Smolenski K.A. Daniel R.M. Morgan H.W. Biochem. J. 1987; 247: 121-133Crossref PubMed Scopus (92) Google Scholar) to several hours for pyrolysin (6Eggen R. Geerling R.A. Watts J. de Vos W.M. FEMS Microbiol. Lett. 1990; 71: 17-20Crossref Google Scholar). Aquifex pyrophilus is a microaerobic eubacterium that grows optimally at 85 °C. This organism is also regarded as the most ancient bacterium because of its autotrophic nature and its deep location in the phylogenetic tree (10Burggraf S. Olsen G.J. Stetter K.O. Woese C.R. Syst. Appl. Microbiol. 1992; 15: 352-356Crossref PubMed Scopus (189) Google Scholar). Several genes homologous to proteases have been identified from the genomic sequence ofAquifex aeolicus (11Deckert G. Warren P.V. Gaasterland T. Young W.G. Lenox A.L. Graham D.E. Overbeek R. Snead M.A. Keller M. Aujay M. Huber R. Feldman R.A. Short J.M. Olsen G.J. Swanson R.V. Nature. 1998; 392: 353-358Crossref PubMed Scopus (960) Google Scholar). In addition, sequence tags similar to protease-like genes such as the lon protease inEscherichia coli allowed the recognition of the signal peptidase and the serine-type protease from the sequence analysis of the genomic DNA of A. pyrophilus (12Choi I.G. Kim S.S. Ryu J.R. Han Y.S. Bang W.G. Kim S.H. Yu G.Y. Extremophiles. 1997; 1: 125-134Crossref PubMed Scopus (11) Google Scholar). The role of the serine-type protease in A. pyrophilus may not be involved in the degradation of extracellular peptides because these compounds are not utilized as carbon or nitrogen sources. To characterize the serine-type protease of A. pyrophilus, we have cloned the entire gene and determined its DNA sequence. The cloned gene belongs to the subtilisin family and is localized in the cell wall fraction of A. pyrophilus as a mature form of 43 kDa. When the gene is transformed into E. coli, it is expressed as active and processed forms, although the expression level is low. This protein shows extreme stability at high temperatures and at a broad range of pH values. Restriction enzymes and T4 DNA ligase were obtained from Promega. Sequencing primers were purchased from Bioneer (Seoul, Korea). A. pyrophilus was obtained from the German Collection of Microorganism and Cell Culture (Deutsch Sammlung von Mikroorganismen, Braunschweig, Germany) and was grown in a modified SME medium (13Stetter K.O. Konig H. Stackebrandt E. Syst. Appl. Microbiol. 1983; 4: 535-551Crossref PubMed Scopus (258) Google Scholar) at 85 °C as described by Huber et al. (14Huber R. Wilharm T. Huber D. Tricone A. Burggraf S. Konig H. Rachel R. Rockinger I. Fricke H. Stetter K.O. Syst. Appl. Microbiol. 1992; 15: 340-351Crossref Scopus (354) Google Scholar). The cells were inoculated into 20 ml of liquid medium in a 120-ml bottle that was filled with 3 bars of hydrogen, carbon dioxide, and oxygen at a ratio of 79.5:20:0.5 and grown for 12 h with moderate shaking. Cells were harvested by centrifugation at 10,000 ×g for 10 min, washed with 10 mm Tris-HCl, pH 8.0, and stored at −80 °C until further usage. The E. coli strain used for plasmid DNA amplification was DH5aH (supE44 ΔlacU169(φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1). E. coli XL1-blue MRA P2 (Δ(mcrA)18 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-l relA1 gyrA96 lac P2 lysogen) was used for λ phage propagation, andE. coli BL21(DE3) (E. coli B F− dcm ompT hsdS(rB- mB-) gal λ(DE3)) was used for protein expression. A. pyrophilus genomic DNA was purified using a genomic DNA purification kit (Qiagen, Hilden, Germany). For the construction of the genomic λ library, 20 μg of genomic DNA from A. pyrophilus was partially digested with HindIII and ligated with 5 μg of λ DASHII digested with HindIII. The ligated DNA was packaged using a packaging kit (Stratagene) and amplified in E. coli MRA (P2) in which only phages containing foreign DNA were propagated. About 4,000 primary plaques were obtained and further amplified. DNAs from λ clones was isolated using a Qiagen λ DNA extraction kit. Standard procedures were used for cloning and sequencing as described by Sambrook et al. (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Southern analysis was performed with an ECL kit from Amersham Pharmacia Biotech (UK). The determination of nucleotide sequence was carried out with an ABI 373 automatic DNA sequencer (Applied Biosystems). The obtained DNA sequences were converted into amino acid sequences in all six possible reading frames and were compared with known proteins in the data base using a sequence comparison program, BLAST (16Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69707) Google Scholar). For multiple sequence alignment between functionally related proteins, the SEQSEE (17Wishart D.S. Boyko R.F. Willard L. Richards F.M. Sykes B.D. Comput. Appl. Biosci. 1994; 10: 121-132PubMed Google Scholar) and MACAW programs (18Schuler G.D. Altschul S.F. Lipman D.J. Proteins. 1991; 9: 180-190Crossref PubMed Scopus (892) Google Scholar) were used. The putative membrane-spanning region was assigned using the TMpred program (19Hofman K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 374: 166-170Google Scholar). Cell paste of A. pyrophilus was suspended in lysis buffer (50 mmTris-HCl, pH 8.0, 20 μg/ml lysozyme) and incubated for 10 min at 37 °C. The cells were sonicated for 2 min using a Branson Sonifier 450 (Branson Ultrasonic Co.). The crude extract was centrifuged for 20 min at 18,000 × g, and the pellet representing the cell wall fraction was recovered. The proteins in the cell wall fraction were extracted with 0.1% SDS at 95 °C for 20 min. The membrane fraction was prepared from the crude extract using serial centrifugation. The crude extract was centrifuged at 10,000 ×g for 10 min, and the supernatant was further centrifuged at 100,000 × g for 1 h. The pellet representing the membrane fraction was resuspended in 50 mm Tris-HCl, pH 8.0. For the preparation of peripheral or integral membrane proteins, the membrane fraction was treated with 1.0 m NaCl or 0.5% Triton and was centrifuged at 100,000 × g for 1 h. of the open reading frame of the cloned protease gene were amplified by the with and primers and The amplified DNA and the were treated with and transformed into E. The transformed cells were grown at 37 °C in medium containing 100 μg/ml with to of Protein expression was by 1 for 3 h. Cells were harvested by centrifugation at 4,000 × 10 min. For the preparation of an to the cloned gene a of the open reading frame representing amino was expressed in E. coli using the as described The expressed from was by used open reading The protein was from the and into a for was obtained For proteins were and to a membrane in 100 mm pH The membrane was treated with a and with in buffer mm Tris-HCl, mm 0.1% pH for 1 h at with protein that to the were using a using the ECL Pharmacia was carried out to the described by PubMed Scopus Google Scholar). The was to 50 ml of the cell wall extract and incubated for 1 h with 50 ml of cells were to the and incubated for 1 h at °C. The was centrifuged at for 10 min, and protease activity in the supernatant was further E. coli cells that expressed the cloned gene were harvested at 4,000 × g for 10 min and resuspended in lysis buffer (50 mm Tris-HCl, pH The cells were by for 2 min, and the fraction was by centrifugation at × g for 20 min. proteins in the crude extract by heat for 20 min at 95 °C, the fraction was a × Pharmacia with 20 mm Tris-HCl, pH 8.0. A from to 1.0 m NaCl in buffer was used to the a or a was used to protease For a were with 0.5% as described by al. (5Cowan D.A. Smolenski K.A. Daniel R.M. Morgan H.W. Biochem. J. 1987; 247: 121-133Crossref PubMed Scopus (92) Google Scholar) with a The of 100 of and of 0.5% in 50 mm pH at 85 °C for min, the was by the of of and the at was measured using a The was carried out using 0.1% as described by and D.E. W.G. Biochem. 1994; PubMed Scopus Google Scholar). were by the Biochem. PubMed Scopus Google Scholar) using as a of the λ clones from the genomic DNA pyrophilus to the sequence tag that a sequence similar to that in serine-type proteases (12Choi I.G. Kim S.S. Ryu J.R. Han Y.S. Bang W.G. Kim S.H. Yu G.Y. Extremophiles. 1997; 1: 125-134Crossref PubMed Scopus (11) Google Scholar). Southern analysis that the sequence tag used as a is in the of the cloned The sequence of the region the sequence tag region has been determined in both 1 In the there is only one open reading frame 100 amino This the serine-type protease consensus sequence in the sequence The nucleotide sequence and amino acid sequence of the are in from the of the consensus sequences were The and sequences at and from the open reading frame are homologous to the and sequences Rev. Biochem. PubMed Scopus Google Scholar). of the putative there are the and that are for of the of them has a sequence in the the sequence at the from the In addition, the amino acid sequences between the and are not proteases. the of 20 amino the has a the gene is in the cell wall fraction the may as a site and the sequence may as a For these we have assigned the as the site for This for a protein of 619 amino and amino acid sequence of the cloned protease gene of A. The is from the at the The putative are in a the catalytic residues in a serine-type The putative signal sequence The sequence is as The amino acid sequence of the cloned gene was compared with gene sequences in data using the BLAST The genes that show strong sequence to the cloned gene are serine-type proteases. has three canonical residues of acid and that the active site of serine-type proteases. acid sequences around these residues are homologous to of proteases The cloned gene has three of sequences at the and and between and 3 The amino at the are to be involved in membrane because the presence of a of amino and them are well to of a signal sequence. The COOH-terminal region from amino acid this region a putative sequence at the COOH-terminal 3 The sequence of amino between and not show sequence with proteins in the data a serine-type protease from A. G.D. K.A. M. J. J. Bacteriol. 1991; PubMed Google and a cell protease from P. G. Siezen R.J. de Vos W.M. J. Biol. Chem. Full Text PDF PubMed Google also have a sequence between the active site and these sequences are not the size of this sequence and the in the gene are To the cloned gene is expressed pyrophilus, analysis was performed the crude extract of A. pyrophilus with an a protein of the cloned A protein from the crude extract of A. pyrophilus was by the with two at and kDa. These are in the cell wall and only are in the fraction These indicate that the cloned gene is expressed in A. pyrophilus as a to the cell and processed to a mature form of 43 kDa. The and are of the When the proteases in the extract of A. pyrophilus were by the at three proteases in the cell wall Among the protease the activity of the with 1 mm phenylmethylsulfonyl most of the protease activity of the and G. that the protease is a serine-type To the protein in the analysis and in the activity is the analysis was The protease with the to the cloned gene and was cells These show that the protein by the cloned gene is the protease in the cell wall The cloned gene has a putative sequence at the To the gene is a membrane the membrane fraction was prepared from the cell extract of A. the protein that with the was in the cell wall a of the protein was in the membrane To the protein is a peripheral or integral membrane extracted protein treated with 1.0 m NaCl or 0.5% Triton was with high not out the protein from the and only a fraction of protein was from the membrane by of extraction by that the protein is not a membrane The protein in the membrane fraction may be because of during cell to the protease from A. pyrophilus were not because of expression and in cell When the gene was expressed in the E. coli BL21(DE3) cell using a the expression level was very and the cells and When the from the at the or were expressed in E. coli, the proteins were expressed at and a the of the region from the at the the and the expressed protein was The expressed protein was not in 6 it was as multiple were in the range of by analysis 6 The size of the expressed protein was reduced heat 6 This that the cloned protease expressed in E. coli To the of the cloned protease, the protein expressed in E. coli was partially purified by and In addition, the thermostability and pH optimum of the protease in the cell wall fraction of A. pyrophilus were also The activity of the protease was at 85 °C and pH and When protease activity of the expressed protein in E. coli was using as a it also a similar to the The activity was measured at °C, and the activity as the temperature The optimum pH for the protease activity was obtained at pH the the protease active in buffer in The stability of the expressed protein was by the in activity at high When the protein was incubated at 85 °C, it was and its half-life was to be h 105 °C, the activity of the protease by for 6 h These indicate that the expressed protein is one of the of the protease expressed The partially purified protease was for its protease activity using as a at pH at 85 °C or temperatures at pH for min. buffer was used at pH 100 mm buffer at pH and 100 buffer at pH of the expressed of the partially purified protease was performed using The protein was incubated at 85 or 105 °C for the and the activity was The cloned protease gene has similar to in the subtilisin the of serine-type proteases 1994; PubMed Scopus Google Scholar). a canonical sequence that has been in a gene of A. pyrophilus J.H. Yu I.G. Ryu J.R. Kim S.H. Han Y.S. Lett. 1997; PubMed Scopus Google Scholar) has not been in the cloned the of the open reading frame was as the site of because it was of the putative The size of the protein was the size the amino acid that the gene was as a protein and processed to a mature subtilisin family proteases are as the containing a signal sequence. The signal sequence was processed the and the sequence was further processed to the mature form H. H. M. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). In the were at both the and of the protein and were during the I. H. T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The amino at the of the open reading frame have in signal sequences H. J. S. von G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus Google Scholar). the signal of the NH2- and COOH-terminal sequences be The between the sequences containing and is size is the size of the mature the residues at the and the residues at the of the form be processed to a is that a putative membrane sequence is at the presumed 3 A sequence at the of a protease that the protein to the membrane has been in the protease P. G. Siezen R.J. de Vos W.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The sequence at the may as a membrane In that the protein may be to the membrane because most of the gene was as a processed form in the cell wall The of the cloned protease in the cell is The protease may not be used for degradation of exogenous because A. pyrophilus is a The role of this protein may be related to its cellular A. pyrophilus a very the that is of the membrane (14Huber R. Wilharm T. Huber D. Tricone A. Burggraf S. Konig H. Rachel R. Rockinger I. Fricke H. Stetter K.O. Syst. Appl. Microbiol. 1992; 15: 340-351Crossref Scopus (354) Google Scholar). The of hyperthermophiles of heat-stable proteins, and it is to the cell from extreme such as high temperature J. M. R. Lupas A. Kellermann J. H. S. K. A. Stetter Baumeister W. J. Mol. Biol. PubMed Scopus Google Scholar). analysis has that a very protease has been to the of the that the J. Lupas A. Kellermann J. Eckerskorn C. Baumeister W. Peters J. Curr. Biol. 1996; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). that the cloned protease is with the proteins of A. pyrophilus, because the protein is to the cell wall the is the protease is involved in the protein degradation or as a of the is The cloned protease is to The half-life of the expressed protein at 105 °C that this is one of the most heat-stable proteases. analysis has that one of the that proteins from hyperthermophiles is bonds or between amino A. R. R. 3: Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, proteins from hyperthermophiles have amino proteins from or A higher of amino is also in the proteins from A. pyrophilus (12Choi I.G. Kim S.S. Ryu J.R. Han Y.S. Bang W.G. Kim S.H. Yu G.Y. Extremophiles. 1997; 1: 125-134Crossref PubMed Scopus (11) Google Scholar). The of and of the cloned protease is This is similar to the in pyrolysin from and is higher proteases from or These residues may be in the of of amino acid of Aquifex protease with of several of of of of of was from P. was from T. was from T. and subtilisin was from in a was from P. was from T. was from T. and subtilisin was from that to the stability of the protease is disulfide are a of cysteine residues in the form of the cloned protein and residues in the mature Most of these cysteine residues form disulfide bonds that the protein because with 1 mm reduced the heat of the protein at 85 °C to 2 disulfide bonds to the high stability of the Among subtilisin family proteases, only a such as have cysteine residues that may form disulfide bonds R.J. Protein Sci. 1997; 6: PubMed Scopus Google Scholar). serine-type proteases from hyperthermophiles not have cysteine residues and J. Lupas A. Kellermann J. Eckerskorn C. Baumeister W. Peters J. Curr. Biol. 1996; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, R.J. Protein Sci. 1997; 6: PubMed Scopus Google Scholar). of stability by of disulfide bonds has been in phage T4 M. G. Nature. PubMed Scopus Google Scholar) or subtilisin H. T. H. M. H. T. J. Biol. Chem. 1990; Full Text PDF PubMed Google of the protein by disulfide bonds is an the serine-type proteases from The of the disulfide bonds that to the stability of the cloned protease is of