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首页> 美国卫生研究院文献>Biochemical Journal >Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits.
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Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits.

机译：产黄顶孢属cefG基因产物的分子特征：天然脱乙酰头孢菌素C乙酰转移酶未加工成亚基。

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摘要

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative alpha- and beta-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.
机译：从产黄曲霉cefG基因的三个框内ATG密码子（Met1，Met46和Met60）开始构建，并在大肠杆菌中表达，分别获得49、44和43 kDa的蛋白质。所有这三种蛋白均显示出脱乙酰头孢菌素C（DAC）乙酰转移酶活性。通过免疫亲和层析将天然产黄曲霉DAC乙酰转移酶纯化至电泳均质。通过在校准的Sephadex G-75 SF或Superose 12（FPLC）色谱柱中过滤显示分子量为50 kDa。纯DAC乙酰基转移酶的N末端是Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu，与从Met1开始的推导氨基酸序列完全匹配。在大肠杆菌中也获得了DAC乙酰基转移酶的推定的α-亚基和β-亚基，但无论是单独还是组合都没有酶活性。免疫印迹（Western）分析显示，50 kDa DAC乙酰基转移酶在72. 96 h的产黄曲霉培养物中显示高蛋白水平，此后急剧下降，但在所有情况下均未发现可检测到的酶加工成亚基的过程。三种不同的产黄曲霉菌株（包括野生型Brotzu菌株和两个生产高头孢菌素的突变株）显示相同的未加工50 kDa DAC乙酰基转移酶。非生产突变体ATCC 20371没有显示DAC乙酰基转移酶蛋白带，但形成了1.4 kb的正常转录本。

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