從嗜甲烷菌Methylcoccus capsulatus(Bath)中分離的微粒型甲烷單加氧酶次單元B，在過去的研究中證實了他與還原態的銅離子有著高度的親合力，但在文獻中根據現有的結晶結構中，只包含三個銅離子。我們認為在微粒型甲烷單加氧酶次單元B上，擁有著E-cluster扮演著傳遞電子的角色，因此探討銅離子與該暴露水層蛋白質間親和鍵結能力以及正確定出銅離子與蛋白質之間的量是很重要的議題。
本篇論文的重點環繞在定點突變及突變後的銅離子差異，我們利用基因工程技術，將微粒型甲烷單加氧酶次單元B透過重組的方式達到有效純化蛋白質的效果；接著利用西方墨點法，透過抗體與MBP蛋白專一性，將其中蛋白質有效的定出內含濃度，並加上ICP-OES之精準的微量金屬定量，計算出蛋白質與銅離子的實際含量；又使用X光吸收光譜，仰賴其靈敏性，相對定量出蛋白質中銅離子之比例；將西方墨點法之精準蛋白質濃度與ICP-OES高準確微量金屬定量再加上X光吸收光譜之靈敏金屬比例分析，計算出互相吻合之定點突變蛋白質銅離子含量。
最後將定點突變蛋白質膜與原始蛋白質膜以及純化後的蛋白質做互相比較，歸納出定點突變與原始蛋白質對於其中內含銅離子的鍵結能力以及其數目多寡的區別。

The PmoB subunit of pMMO from Methylococcus capsulatus (Bath) exhibits strong affinity towards CuI. The C-terminal aqueous sub-domain of PmoB is capable of sequestering ca. 10 reduced coppers, and it has been proposed that these CuI serve as a reservoir of reducing equivalents for methane oxidation in the holo enzyme. To confirm the high copper affinity in the full subunit, we have translocated and over-expressed the PmoB fused with the maltose-binding protein (MBP) into a number of E. coli strains. The recombinant PmoBs are verified by Western blotting of the antibodies against both MBP and copper-enriched PmoB. Using ICP-OES to confirm metal content. The recombinant PmoBs will show some information by X-ray absorption spectroscopy. When a bacterial strain BCRC®50305 tolerant against CuCl2 up to 3.1 mM is grown with the pmoB gene in 1.0 mM CuII, the E. coli behaves like M. capsulatus (Bath) cultured under high copper stress with abundant membrane accumulation and high CuI content. These results suggest that both M. capsulatus (Bath) and E. coli variants bear similar capacities for membrane production and incorporation of cellular CuI under copper stress. With control over copper homeostasis, the E. coli variants assist the pMMO B subunit to assemble into the cytoplasmic membrane and achieve maturation.

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