吳郭魚MyoD / 斑馬魚 Myf5 肌肉特異性轉錄因數重組蛋白的表現、純化及DNA親和力的研究

Abstract

胚胎發生過程中骨骼肌的生成，是經由許多因數協調控制才能完成，其中包括肌肉特異性轉錄因數群 ( MRFs ) 的四位成員 - Myf5、MyoD、Myogenin 及 MRF4，這些蛋白質的表現具有時間、位置的侷限性，並且會與基因上游?動子區域的 E- box ( CANNTG ) 結合，進而調控肌肉特異性基因的表現。 肌肉特異性轉錄因數群均為核蛋白，不只量少，且其表現是動態的、瞬間的，蛋白質萃取不易，造成研究上的困難。因此，瞭解其分子結構以便獲得重組蛋白，將有助對於其生物特性、蛋白質結構及 molecule interaction 的相關研究，更亦可用以製造抗體。本實驗先以吳郭魚為實驗動物，將吳郭魚 MyoD 與目前所知魚類 zebrafish、carp、rainbow trout 1、rainbow trout 2 的 MyoD 胺基酸序列做比較，相似度分別為 64.3、64.1、62.6、62.4％。而在 basic helix-loop-helix domains 則幾乎完全相同。以 His•Tag system，利用 Escherichia coli 製造吳郭魚 MyoD 與 Histidine tag 融合的蛋白（分子量約35kDa），使用 Ni2+-NTA spin column 進行純化，得到 0.3 mg/ml，純度 95％以上的吳郭魚重組蛋白。純化後的蛋白，可以為老鼠 MyoD 的多株抗體所辨識。將純化後的蛋白進行 electrophoretic mobility shift assay ( EMSA )，發現吳郭魚 MyoD 重組蛋白與 E-box 具有結合能力。同樣地，以 His•Tag system，利用 E . coli 製造斑馬魚 Myf5 與 Histidine tag 融合的蛋白（分子量約 32 kDa），以 Ni2+-NTA agarose 自行packing column 進行純化，得到 5 mg/ml，純度 95％以上的斑馬魚 Myf5 重組蛋白。純化後的 Myf5 重組蛋白，亦有與 E-box具有結合能力。因此，證明簡單的原核細胞即可製造具有生物活性的 MyoD 及 Myf5 重組蛋白。

Skeletal muscle development has become a paradigm for understanding the mechanisms that control cell specification and differentiation during embryogenesis. Myogenesis is regulated by a family of four transcription factors (Myf5, MyoD, myogenin, and MRF4) that share a common dimerization and DNA binding domain, the basic helix-loop-helix (bHLH) motif, which capable to bind a specific sequence of DNA fragment, i.e., E-box(CANNTG). Those proteins are nuclear proteins, and the temporal-spatial patterns are restricted. In the experiment I, as the tilapia MyoD be the subject, using the Clustalw program to finish the tilapia MyoD sequence analysis, tilapia MyoD shared sequence identities of 64.3, 64.1, 62.6 and 62.4％ with those of zebrafish, carp, and two rainbow trout, respectively. Results from a molecular phylogenic tree assay showed that the tilapia MyoD was more closely related to those of other fishes than of higher vertebrates. We construct the expression vector of tilapia MyoD, and use Escherichia coli, a His•tag system, and a Ni2+-NTA spin column, we purified ~35 kDa recombinant tilapia MyoD. Results from an electrophoretic mobility shift assay demonstrated that the purified E. coli-produced tilapia MyoD was capable of binding to the DNA fragment sequence CA(C/T)(C/A)TG. In the experiment II, as the zebrafish Myf5 be the following subject, the Clustalw suquence analysis indicates that zebrafish Myf5 shared sequence identities of 72.4, 63.3％ with zebrafish MyoD, myogenin, respectively. Results from a molecular phylogenic tree assay showed that the Myf5 is so close between the species, since, we thought that Myf5 may has functional conserved. We construct the expression vector of zebrafish Myf5, and use E. coli, a His•tag system, and a Ni2+-NTA agarose column, we purified ~32 kDa recombinant tilapia MyoD. Results from an electrophoretic m obility shift assay demonstrated that the purified E. coli-produced zebrafish Myf5 was capable of binding to the DNA fragment sequence CACCTG.