於大腸桿菌中利用σ32因子與目標蛋白質共表達來改善重組酵素蛋白質的可溶性及活性

Improve solubility and activity of a recombinant enzyme protein by co-expressing sigma 32 factor in Escherichia coli

許紹彥(Hsu, Shaoyen)

指導教授 ： 李文乾

國立中正大學/工學院/化學工程學系/碩士(2012年)

摘要

本研究使用大腸桿菌BL21過量表現融合酵素蛋白質N-乙醯-D-神經胺糖酸醛縮酶(Neu5Ac aldolase)，其中，酵素之N端連接麩胱苷肽-S轉移酶蛋白(GST, Glutathione S-transferase)，C端融合五個精胺酸(5R, 5 Arginine)形成雙標誌酵素蛋白質。實驗上利用基因重組方法將實驗室現有的菌種E. coli BL21 pGEX-2TK-nana-5R加入新切位成為E. coli BL21 pGEX-2TK-nana-5R-XhoI(簡稱XhoI，控制組)，再將表現σ32因子的基因(rpoH)嵌入pGEX-2TK質體中，接於Neu5Ac aldolase序列後面以shine-dalgrno ribosome binding site連結成為E. coli BL21 pGEX-2TK-nana-5R-rpoH(簡稱rpoH，實驗組)，額外比較一株實驗組的雙質體菌種E. coli BL21 pGEX-2TK-nana-5R & pBAD33-rpoH(簡稱dual，實驗組)，分別加入異丙基-D-硫代半乳糖(IPTG)或阿拉伯糖(arabinose)誘導後共表達，進一步去探討蛋白質體變化以及目標酵素活性與溶解度。嵌入rpoH基因主要是希望σ32因子以過量表達的方式代替熱休克這類的應力條件來間接促進下游的熱休克蛋白質(輔酶)表現，藉此方法以補強大腸桿菌表現外源蛋白時，會產生包涵體缺點的不足進而使外源蛋白的溶解度及活性能有所提升，使得工業化量產酵素時，能夠有效地降低製程過程中的成本。
本實驗研究以二維膠體電泳探討上述三株菌種誘導前後的熱休克蛋白與σ32因子之間的蛋白質體變化，發現XhoI、rpoH以及dual之菌種於誘導三小時後，小熱休克蛋白ibpA/B分別有顯著上升(p-value < 0.05)； rpoH比起XhoI於誘導三小時後，小熱休克蛋白ibpA/B分別有顯著下降(p-value < 0.05)。
　　在西方點墨法實驗中顯示XhoI在誘導後1、2及3小時比起未誘導時，σ32因子的表現量提升了3.5、2.1以及2.1倍，dual則提升了12.6、11以及12.8倍，rpoH提升了15.3、16.4以及19.1倍；在溶解度方面，rpoH以及dual比起XhoI於誘導1小時的溶解度有明顯上升至27%以及10% ( p-value < 0.05)，但隨著誘導時間增長而下降。
　　在活性實驗方面，顯示rpoH比起XhoI在誘導後1、2及 3小時的全細胞比活性分別提升了1.6、1.42、以及1.41倍，dual比起XhoI提升了3.23、2.63以及2.29倍；菌體破萃後上清液比活性rpoH比起XhoI提升了1.6、3以及2.5倍，dual比起XhoI則是提升了2、1.5以及1.7倍；而剩下不溶體回溶液的比活性rpoH比起XhoI則提升了1.4、1.8、2.4倍，dual則比XhoI提升了5、4以及2.7倍。
　　因此本實驗研究證實含有rpoH基因共表達之菌株，所生產出來的目標酵素蛋白質有較好的溶解度以及活性，與一開始的假設一致。

關鍵字

sigma 32因子 ； 熱休克轉錄因子 ； N-乙醯-D-神經胺糖酸醛縮酶 ； 共表達 ； 包涵體 ； 構形品質

並列摘要

We used Escherichia coli BL21 overexpressing recombinant enzyme protein, N-acetyl-D-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R or Neu5Ac aldolase), this enzyme protein overexpressed with glutathione S-transferase (GST, Glutathione S-transferase) and polyionic peptide (5R, 5 Arginine) tags became two tags fusion enzyme protein. By genetic engineering technique, we add a new cutting site into the existed E. coli strain (E. coli BL21 pGEX-2TK nana5R), to become E. coli BL21 pGEX-2TK nana5R-XhoI (abbr. XhoI). At the same time we add σ32 factor gene (rpoH) into pGEX-2TK with shine-dalgrno ribosome binding site, right after Neu5Ac aldolase sequence, to become E. coli BL21 pGEX-2TK nana5R-rpoH (abbr. rpoH). During E. coli culture, we add Isopropyl β-D-1-thiogalactopyranoside (IPTG) or arabinose to induce these engineered enzymatic protein expression. Compare with E. coli BL21 pGEX-2TK nana5R and pBAD33 rpoH (abbr. dual, control group), we studied for the changes of proteomics and the activity or solubility of target enzymatic protein. We add rpoH gene to overexpressing σ32 factor, that offsets the conditions of heat-shock. In this condition, E. coli will express heat-shock proteins to improve the defects of low solubility and activity of overexpressed proteins while inclusion body forming. This way will reduce the cost of artificial protein production.
　　Then we extract the protein of E. coli of each group. Through 2-dimensional SDS-PAGE to study the proteomics changes of heat-shock proteins and σ32 factor. The results indicated that XhoI, rpoH and dual groups showed a significant increase of ibpA/B after 3 hours of IPTG or arabinose induction compare with control group (p < 0.05). And a significant decrease of ibpA/B protein in rpoH group compare with XhoI group after 3 hours of IPTG induction (p < 0.05).
　　In western blot, that also indicated σ32 protein increased 3.5 times, 2.1 times and 2.1times in XhoI group after 1, 2, 3 hours of IPTG induction compare with 0 hour. And σ32 protein increased 12.6 times, 11 times and 12.8times in dual group after 1, 2, 3 hours of IPTG and arabinose induction compare with 0 hour. In rpoH group, σ32 protein increased 15.3 times, 16.4 times and 19.1 times after 1, 2, 3 hours of IPTG or arabinose induction compare with 0 hour. The solubility of Neu5AC aldolase had a significant 27% increase in rpoH group compare to XhoI group after 1 hour of IPTG induction, but decrease with induction time. The solubility of Neu5AC aldolase had a significant 10% increase in dual group compare to XhoI group after 1 hour of IPTG and arabinose induction, but decrease with induction time.
　　Another results showed that whole cell activity in rpoH group increased 1.6 times, 1.42 times and 1.41 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And the whole cell activity in dual group also increased 3.23 times, 2.63 times and 2.29 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group. The specific activity of supernatant after lysis in rpoH group increased 1.6 times, 3 times and 2.5 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And the specific activity of dual group also increased 2 times, 1.5 times and 1.7 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group. The specific activity in the rest insoluble in rpoH group increased 1.4 times 1.8 times and 2.4 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And in dual group also increased 5 times, 4 times and 2.7 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group.

並列關鍵字

sigma 32 ； heat shock transcription factor ； N-acetyl-D-neuraminic acid aldolase ； co-expression ； inclusion body ； conformational quality

參考文獻

蘇遠志、黃世佑，微生物化學工程學(2006)

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