The gene of MTSase (maltooligosyltrehalose synthase) from \%Sulfolobus acidocaldarius\% ATCC49426 was amplified by PCR.The primers were designed according to the published sequence of homologous gene from \%Sulfolobus...The gene of MTSase (maltooligosyltrehalose synthase) from \%Sulfolobus acidocaldarius\% ATCC49426 was amplified by PCR.The primers were designed according to the published sequence of homologous gene from \%Sulfolobus acidocaldarius \%ATCC33909.This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220\|GT was transformed to \%E.coli\% DH5α.The activity of recombinant enzyme was about 10u/g(wet cell).In order to improve the expression level of target protein,some nucleotides in the 3′ and 5′ of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0.As a result,the activity of recombinant enzyme increase to 19.8u/g(wet cell).Then,the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain.But it took little effect.展开更多
To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recomb...To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polBl（Saci_1537） and polB3（Saci_0074） possessed DNA polymerase and 3＇ to 5＇ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV（Saci_0554） was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.展开更多
文摘The gene of MTSase (maltooligosyltrehalose synthase) from \%Sulfolobus acidocaldarius\% ATCC49426 was amplified by PCR.The primers were designed according to the published sequence of homologous gene from \%Sulfolobus acidocaldarius \%ATCC33909.This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220\|GT was transformed to \%E.coli\% DH5α.The activity of recombinant enzyme was about 10u/g(wet cell).In order to improve the expression level of target protein,some nucleotides in the 3′ and 5′ of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0.As a result,the activity of recombinant enzyme increase to 19.8u/g(wet cell).Then,the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain.But it took little effect.
基金Supported by the National Natural Science Foundation of China（No.31371260） and the Natural Science Foundation of Shanghai City, China（No. 12ZR1413700）. We greatly appreciate Professor Sonja-Verena Albers for kindly providing the strain of S. acidocaldarius.
文摘To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polBl（Saci_1537） and polB3（Saci_0074） possessed DNA polymerase and 3＇ to 5＇ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV（Saci_0554） was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.
