Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus
Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC126...
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| Veröffentlicht in: | Bioinformation 2013-01, Vol.9 (4), p.169-173 |
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| creator | Lakshmi, Hanumanthu Prasanna Yeswanth, Sthanikam Prasad, Uppu Venkateswara Vasu, Dudipeta Swarupa, Vimjam Kumar, Pasupuleti Santhosh Narasu, Mangamoori Lakshmi Krishna Sarma, Potukuchi Venkata Gurunadha |
| description | Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis. |
| doi_str_mv | 10.6026/97320630009169 |
| format | Article |
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The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.</description><identifier>ISSN: 0973-2063</identifier><identifier>ISSN: 0973-8894</identifier><identifier>EISSN: 0973-2063</identifier><identifier>DOI: 10.6026/97320630009169</identifier><identifier>PMID: 23519063</identifier><language>eng</language><publisher>Singapore: Biomedical Informatics</publisher><subject>Escherichia coli ; Hypothesis ; Staphylococcus aureus</subject><ispartof>Bioinformation, 2013-01, Vol.9 (4), p.169-173</ispartof><rights>2013 Biomedical Informatics 2013</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c353t-f2496592b477bec433257fd2438f26c06f7630b1faf2d97c6f67f99cb8035de73</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602885/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602885/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23519063$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Kangueane, P</contributor><creatorcontrib>Lakshmi, Hanumanthu Prasanna</creatorcontrib><creatorcontrib>Yeswanth, Sthanikam</creatorcontrib><creatorcontrib>Prasad, Uppu Venkateswara</creatorcontrib><creatorcontrib>Vasu, Dudipeta</creatorcontrib><creatorcontrib>Swarupa, Vimjam</creatorcontrib><creatorcontrib>Kumar, Pasupuleti Santhosh</creatorcontrib><creatorcontrib>Narasu, Mangamoori Lakshmi</creatorcontrib><creatorcontrib>Krishna Sarma, Potukuchi Venkata Gurunadha</creatorcontrib><creatorcontrib>Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, India 517 507</creatorcontrib><title>Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus</title><title>Bioinformation</title><addtitle>Bioinformation</addtitle><description>Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.</description><subject>Escherichia coli</subject><subject>Hypothesis</subject><subject>Staphylococcus aureus</subject><issn>0973-2063</issn><issn>0973-8894</issn><issn>0973-2063</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><recordid>eNqFkc1P3DAQxa0KVCj02iPykUMD_kjs-FKpWtEPCakH4Gw5znjjNmsHO1lBz_3D69VSBKfKB4-ef36amYfQB0ouBGHiUknOiOCEEEWFeoOOSVGqnXTwoj5C73L-SUhNpWzeoiPGG6qKfoz-rMYYfFh_xPAwJcjZx4BN6LEdTDJ2huR_m3knRofX42LjLx9MBryGANiHbRy30JcCzwPsgQyVqPA0xDwNZgbsYtrsLQp1M5tpeByjjdYuGZslwZJP0aEzY4b3T_cJuvtydbv6Vl3_-Pp99fm6srzhc-VYrUSjWFdL2YGtOWeNdD2reeuYsEQ4WTbRUWcc65W0wgnplLJdS3jTg-Qn6NPed1q6DfQWwpzMqKfkNyY96mi8fv0S_KDXcat52XXbNsXg_MkgxfsF8qw3PlsYRxMgLllTrpRivC3n_yhVtGWK7dq62KM2xZwTuOeOKNG7lPXrlMuHs5dzPOP_YuV_Ad6xpRw</recordid><startdate>20130101</startdate><enddate>20130101</enddate><creator>Lakshmi, Hanumanthu Prasanna</creator><creator>Yeswanth, Sthanikam</creator><creator>Prasad, Uppu Venkateswara</creator><creator>Vasu, Dudipeta</creator><creator>Swarupa, Vimjam</creator><creator>Kumar, Pasupuleti Santhosh</creator><creator>Narasu, Mangamoori Lakshmi</creator><creator>Krishna Sarma, Potukuchi Venkata Gurunadha</creator><general>Biomedical Informatics</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7QL</scope><scope>7QO</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20130101</creationdate><title>Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus</title><author>Lakshmi, Hanumanthu Prasanna ; Yeswanth, Sthanikam ; Prasad, Uppu Venkateswara ; Vasu, Dudipeta ; Swarupa, Vimjam ; Kumar, Pasupuleti Santhosh ; Narasu, Mangamoori Lakshmi ; Krishna Sarma, Potukuchi Venkata Gurunadha</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c353t-f2496592b477bec433257fd2438f26c06f7630b1faf2d97c6f67f99cb8035de73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Escherichia coli</topic><topic>Hypothesis</topic><topic>Staphylococcus aureus</topic><toplevel>online_resources</toplevel><creatorcontrib>Lakshmi, Hanumanthu Prasanna</creatorcontrib><creatorcontrib>Yeswanth, Sthanikam</creatorcontrib><creatorcontrib>Prasad, Uppu Venkateswara</creatorcontrib><creatorcontrib>Vasu, Dudipeta</creatorcontrib><creatorcontrib>Swarupa, Vimjam</creatorcontrib><creatorcontrib>Kumar, Pasupuleti Santhosh</creatorcontrib><creatorcontrib>Narasu, Mangamoori Lakshmi</creatorcontrib><creatorcontrib>Krishna Sarma, Potukuchi Venkata Gurunadha</creatorcontrib><creatorcontrib>Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, India 517 507</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Bioinformation</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lakshmi, Hanumanthu Prasanna</au><au>Yeswanth, Sthanikam</au><au>Prasad, Uppu Venkateswara</au><au>Vasu, Dudipeta</au><au>Swarupa, Vimjam</au><au>Kumar, Pasupuleti Santhosh</au><au>Narasu, Mangamoori Lakshmi</au><au>Krishna Sarma, Potukuchi Venkata Gurunadha</au><au>Kangueane, P</au><aucorp>Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, AP, India 517 507</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus</atitle><jtitle>Bioinformation</jtitle><addtitle>Bioinformation</addtitle><date>2013-01-01</date><risdate>2013</risdate><volume>9</volume><issue>4</issue><spage>169</spage><epage>173</epage><pages>169-173</pages><issn>0973-2063</issn><issn>0973-8894</issn><eissn>0973-2063</eissn><abstract>Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). 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| source | Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; PubMed Central Open Access |
| subjects | Escherichia coli Hypothesis Staphylococcus aureus |
| title | Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus |
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