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首页> 外文期刊>Acta Biochimica Polonica >Cloning, sequence, expression and characterization of human beta-mannosidase
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Cloning, sequence, expression and characterization of human beta-mannosidase

机译：人β-甘露糖苷酶的克隆，序列，表达和表征

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摘要

P-Mannosidase (EC 3.2.1.25, MANB) dissects the non-reducing end of N-linked mannose moieties of glycoproteins in eukaryotic cells. The human P-mannosidase gene was amplified by RTPCR, cloned and sequenced. The DNA sequence was compared with reported human beta-mannosidase DNA sequence and sixteen nucleotide differences were found. The deduced amino-acid sequence showed that seven codons coded the same amino acids and nine codons coded different amino acids with reference to nucleotide substitution positions but did not affect recombinant MANB enzyme activity. No splice mutation was observed after comparison with reported MANB DNA sequences. A 75% homology of deduced amino-acid sequence was observed with mouse, goat and bovine beta-mannosidase amino-acid sequences. The cloned beta-mannosidase gene was subcloned into pET22b+ and pET28a+ expression vectors to transform the 131,21-codon plus cells for expression of recombinant MAN22 and MAN28 enzymes, respectively. The optimized conditions for overexpression of recombinant beta-mannosidase enzyme were induction with 1 mM IPTG for 12 h at 37 degrees C. The expressed beta-mannosidase enzyme was purified to homogeneity by a combination of DEAE-ion exchange and size exclusion chromatography. The molecular mass of MAN22 and MAN28 enzymes is 97 kDa by SDS/PAGE and is confirmed by Western blot analysis. The recombinant enzymes are active at 37 degrees C and at pH 5.0 and showed activity with beta-nitrophenyl-beta-D-mannopyranoside and not with p-nitrophenyl-alpha-D-mannopyranoside. The K-m value of enzymes was 2.53 mM. The enzyme activity was inhibited by Zn2+, Co2+, Cu2+, Pb2+, Ag1+, iodoacetate, SDS, DMF, DMSO and ethanol. Fe3+, Ca2+ Mg2+, Mn2+, Triton X-100 and PMSF did not inhibit the enzyme activity. Northern blot analysis showed a transcript of about 3.7 kb in all cells and tissues studied. This is the first report on the expression and characterization of recombinant human MANB enzyme.
机译：P-甘露糖苷酶（EC 3.2.1.25，MANB）剖析了真核细胞中糖蛋白的N-连接甘露糖部分的非还原端。通过RTPCR扩增人P-甘露糖苷酶基因，克隆并测序。将DNA序列与报道的人β-甘露糖苷酶DNA序列进行比较，发现十六个核苷酸差异。推导的氨基酸序列表明，参照核苷酸取代位置，七个密码子编码相同的氨基酸，而九个密码子编码不同的氨基酸，但不影响重组MANB酶的活性。与报告的MANB DNA序列比较后未观察到剪接突变。观察到与小鼠，山羊和牛β-甘露糖苷酶氨基酸序列的推定氨基酸序列具有75％的同源性。将克隆的β-甘露糖苷酶基因亚克隆到pET22b +和pET28a +表达载体中，以分别转化131,21-密码子+细胞，以表达重组MAN22和MAN28酶。重组β-甘露糖苷酶过表达的最佳条件是在37℃下用1 mM IPTG诱导12 h。通过DEAE离子交换和尺寸排阻色谱法将表达的β-甘露糖苷酶纯化至均质。通过SDS / PAGE，MAN22和MAN28酶的分子量为97kDa，并通过蛋白质印迹分析证实。所述重组酶在37℃和pH 5.0下具有活性，并且显示出与β-硝基苯基-β-D-甘露吡喃糖苷而不具有对-硝基苯基-α-D-甘露吡喃糖苷的活性。酶的K-m值为2.53mM。酶活性受到Zn2 +，Co2 +，Cu2 +，Pb2 +，Ag1 +，碘乙酸盐，SDS，DMF，DMSO和乙醇的抑制。 Fe3 +，Ca2 +，Mg2 +，Mn2 +，Triton X-100和PMSF均不抑制酶的活性。 Northern印迹分析显示在所有研究的细胞和组织中约3.7kb的转录本。这是有关重组人MANB酶的表达和表征的第一份报告。

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