咖啡α-半乳糖苷酶基因的克隆与表达研究
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摘要
α-半乳糖苷酶(α-D-galactosidase,α-Gal,EC 3.2.1.22)是一种外切糖苷酶，广泛存在于生物界中。不同来源的α-Gal分子量介于28000～370000之间，有单体、二聚体、三聚体与四聚体形式。由于某些来源的α-Gal具有血型转换的特殊功能，对该酶的研究越来越多，从咖啡来源的α-Gal在血型转换方面的效果表现最好，目前，采用生物工程手段进行研究已开始为科枝界所关注。编码小粒种咖啡(Coffea arabica)α-Gal的cDNA约为1384bp，编码377个氨基酸，而成熟肽编码区的cDNA约为1089bp，编码363个氨基酸。
本研究分离克隆了大粒种咖啡(Coffea liberica)与中粒种咖啡(Coffea canephora)的α-Gal成熟肽编码区的cDNA，而后利用二种微生物表达系统进行重组发酵表达的研究，研究结果如下：
1．利用试剂盒的改进方法，简便、快捷而有效地提取到质量良好的热带作物咖啡的总RNA，全过程仅需2h。
2．首次从大粒种咖啡(C.liberica)与中粒种咖啡(C.canephora)中，克降到α-Gal cDNA的成熟肽编码区α-Gal-D cDNA和α-Gal-2 cDNA。克隆到的α-Gal-DcDNA编码区为1089bp，与已发表的小粒种咖啡成熟肽编码序列1080bp有98.7％同源性，共有14处碱基发生变化，氨基酸发生突变的布8个；克隆到的α-Gal-Z cDNA编码区也为1089bp，与已发表的小粒种咖啡成熟肽编码序列1089bp有99.27％同源性，共有8处碱基发生变化，氨基酸发生突变的有4个。
3．将克隆到的大粒种与中粒种咖啡的α-Gal基因，用巴斯德毕赤氏酵母Pichia pastoris表达载体pPICZαA(分泌甲醇诱导型)和pGAPZαA(分泌组成型)，成功地构建了4个酵母表达载体：pPICZαA／Gal-Z,pPICZαA／Gal-D,pGAPZαA／Gal-Z,pGAPZαA／Gal-D。
4．将克隆到的大粒种咖啡的α-Gal基因，用大肠杆菌Escherichia cali的分泌型表达载体pET-22b(+)，成功地构建了1个大肠杆菌表达载体：pET-22／Gal-D。
5．对重组P.pastoris工程菌pPICZαA／Gal-D／GS115、pPICZαA／Gal-Z／GS115、pGAPZαA／Gal-D／GS115进行了摇瓶发酵表达，对发酵产物进行了SDS-PAGE电泳，得到一条约为40KD的主条带，确认了rα-Gal-D与rα-Gal-Z均得到了表达；并对表达产物进行了酶活性检测，结果证明了rα-Gal-D与rα-Gal-Z均具有活性。
6．附消卜积的把）院发酵采用甲醇诱导到我休p卜1＊Z。。A叫，分泌发达的r。－
Gal－D的酶活在481。时为 19．11（U／Inl）高于ro－Gal－Z在48llDI；J为 15．93（［J／Inl），
蛋一质分泌品却是 r。－Ga1Z为 692．5（mg／卜）高下 r ala卜D为 504．5（lllg／卜）。说
明咖，仰不同种来源的。－G。1共同在同一表达载体中，表达员有差异：八分泌表达中，
蛋白质分泌最高，酶活不一定就高，二者不成Ill比。实验中对r。。刀。卜D酶活检测
在 108 11时，其活性可高达 48．22（U／1ill），而 IO－GOIL则为 18．18（U／：11I）。
7．同一个咖l咋种来源的a－Gal基冈，在不同的表达政体中表达乌山有差异，甲
醇诱导刊载体 pP！CZ O A优下组成刑载体 pGAPZ。A。
8．对产paslorj，1程苗pPICZ。A／GalD／GS］15讲行J十发阶规发附表达，对
发酵产’物进行了S＊巳＊AOE丁匕泳，得到一条约为40KD的条带，确认了r a刁a卜D
得到了表达；并对产物进行酶活性检测，证明了 r a－Gal－D只有酶活性。
9．对Ecoli工程菌PET、22／GalD／BL21进行了摇瓶发酵表达，对发酵产物进行
了SDS－PAGE电泳，目的条带区分不明显：并对发酵产物进行了酶活性检测，只有
做最酶活性，说明该表达载体与宿主菌可能不适于。一日。卜D基闭的表达。
a-D-galactosidase ( a-Gal, R. C. 3. 2. 1. 22 ) i s an exo-glycosidaso, widely present in the bio-resources. The enzyme isolated from coffee beans has been well characterized and it has high activity in hydrolyzing the terminal a -Gal residues from glycoconjugates on human blood group B erythrocytes to demonstrate group 0 serotype. a -Gal from coffee bean has been successf u 11 y used i n the serologicl conversion of red blood cells in clinical studies of blood transfusion. The molecular weight of different sources of a-Gal is between 28 000-370 000, and the molecular weight of a-Gal from coffee bean(Coffea arabicd) is 41 000. The whole Tenth cDNA of a -Gal from coffee bean(C. arabicd) is 1384bp and that of the mat peptides cDNA is 1089bp.
The results of this study are as follows:
l.A Simple, fast, and efficiency method for extracting of total RNA from coffee bean has been used by RNA extracting Kit which were improved in this paper.
2. Two mat_peptides cDNA encoding n -Gal have been cloned by RT-PCR from coffee bean ( C. liberica & C. canephora ) . The mat_peptidcs cDNA are 1089bp, encoded 364 amino acid(AA). The cDNA from C. liberica is 98.7% homologous to the published C. arabica sequence. It has been found that 14bp of DNA sequence or 8 AA of protein sequence were changed. The cDNA of u -Gal gene from C. canephora is 99.27% homologous to the published C. arabica sequence,while 8bp of DNA sequence or 4 AA of protein sequence were changed.
3. Four expression vectors, pPICZ a A/Gal-Z, pPICZ a A/Ga1-D, pGAPZ a A/Gal-Z, pGAPZ a A/Gal-D have been constructed successfully with the a-Gal gene from coffee bean and the vectors, pPICZ a A and pGAPZ a of Pichia pasloris.
4. One Excherichia coli expression vector, pFT-22/Gal-D has been constructed with the a -Gal gene isolated from C. liberica and the E coli secretion vector pET-22b ( + ) .
5. The three constructed vectors pPICZ a A/Gal-Z, pPICZa A/Gal-D, pGAPZ a A/Gal-D were transferred into P. pastoris stain GS115. The shake-flask expression with recombinant strain of P. pastoris, pPICZ a A/Gal-Z/GS115, pPICZ a A/Gal-D/GS115, pGAPZ a A/Gal-D/GSl15; the fermentation expression with recombinant strain of P. pastoris , pPICZ a A/Gal-D/GSl15 have been made. The products of shake-flask and fermentation expression showed a main band of 40KD on SDS-PAGE.
6. The enzyme assay showed the high enzyme activity of r a -galactosidase when using pPICZ a A vector.The enzyme activity from C. liberica was 19.11 (U/ml)at 48 h and up to 48.22( U/ml )at 108 h. the en/yme activity from C. cancphora was 15.93 (U/ml) at 48 h and up to 18.18 (U/ml) at 108 h.
7. There were some differences between the r a -Gal quantities of expression with different vectors, the methanol inducing vector, pPICZ a A was better than that of the constitute vector pGAPZ a A.
8. The constructed vector pPICZa A/Gal-D has been transferred into P. pastoris stain GS115, followed by fermentation, a single protein band of 40 KD showed on SDS-PAGE. The enzyme assay showed the activity of a -galactosidase.
9. The constructed vector pET-22/Gal-D was transferred into E. coli stain DL21(DE3)pLysS, followed by shake-flask expression. There was not obviously objective protein band has been seen on SDS-PAGE. The enzyme assay showed a light activity of a -Gal. The result showed that the a -Gal gene was not well expression in this expression system.
本研究分离克隆了大粒种咖啡(Coffea liberica)与中粒种咖啡(Coffea canephora)的α-Gal成熟肽编码区的cDNA，而后利用二种微生物表达系统进行重组发酵表达的研究，研究结果如下：
1．利用试剂盒的改进方法，简便、快捷而有效地提取到质量良好的热带作物咖啡的总RNA，全过程仅需2h。
2．首次从大粒种咖啡(C.liberica)与中粒种咖啡(C.canephora)中，克降到α-Gal cDNA的成熟肽编码区α-Gal-D cDNA和α-Gal-2 cDNA。克隆到的α-Gal-DcDNA编码区为1089bp，与已发表的小粒种咖啡成熟肽编码序列1080bp有98.7％同源性，共有14处碱基发生变化，氨基酸发生突变的布8个；克隆到的α-Gal-Z cDNA编码区也为1089bp，与已发表的小粒种咖啡成熟肽编码序列1089bp有99.27％同源性，共有8处碱基发生变化，氨基酸发生突变的有4个。
3．将克隆到的大粒种与中粒种咖啡的α-Gal基因，用巴斯德毕赤氏酵母Pichia pastoris表达载体pPICZαA(分泌甲醇诱导型)和pGAPZαA(分泌组成型)，成功地构建了4个酵母表达载体：pPICZαA／Gal-Z,pPICZαA／Gal-D,pGAPZαA／Gal-Z,pGAPZαA／Gal-D。
4．将克隆到的大粒种咖啡的α-Gal基因，用大肠杆菌Escherichia cali的分泌型表达载体pET-22b(+)，成功地构建了1个大肠杆菌表达载体：pET-22／Gal-D。
5．对重组P.pastoris工程菌pPICZαA／Gal-D／GS115、pPICZαA／Gal-Z／GS115、pGAPZαA／Gal-D／GS115进行了摇瓶发酵表达，对发酵产物进行了SDS-PAGE电泳，得到一条约为40KD的主条带，确认了rα-Gal-D与rα-Gal-Z均得到了表达；并对表达产物进行了酶活性检测，结果证明了rα-Gal-D与rα-Gal-Z均具有活性。
6．附消卜积的把）院发酵采用甲醇诱导到我休p卜1＊Z。。A叫，分泌发达的r。－
Gal－D的酶活在481。时为 19．11（U／Inl）高于ro－Gal－Z在48llDI；J为 15．93（［J／Inl），
蛋一质分泌品却是 r。－Ga1Z为 692．5（mg／卜）高下 r ala卜D为 504．5（lllg／卜）。说
明咖，仰不同种来源的。－G。1共同在同一表达载体中，表达员有差异：八分泌表达中，
蛋白质分泌最高，酶活不一定就高，二者不成Ill比。实验中对r。。刀。卜D酶活检测
在 108 11时，其活性可高达 48．22（U／1ill），而 IO－GOIL则为 18．18（U／：11I）。
7．同一个咖l咋种来源的a－Gal基冈，在不同的表达政体中表达乌山有差异，甲
醇诱导刊载体 pP！CZ O A优下组成刑载体 pGAPZ。A。
8．对产paslorj，1程苗pPICZ。A／GalD／GS］15讲行J十发阶规发附表达，对
发酵产’物进行了S＊巳＊AOE丁匕泳，得到一条约为40KD的条带，确认了r a刁a卜D
得到了表达；并对产物进行酶活性检测，证明了 r a－Gal－D只有酶活性。
9．对Ecoli工程菌PET、22／GalD／BL21进行了摇瓶发酵表达，对发酵产物进行
了SDS－PAGE电泳，目的条带区分不明显：并对发酵产物进行了酶活性检测，只有
做最酶活性，说明该表达载体与宿主菌可能不适于。一日。卜D基闭的表达。
a-D-galactosidase ( a-Gal, R. C. 3. 2. 1. 22 ) i s an exo-glycosidaso, widely present in the bio-resources. The enzyme isolated from coffee beans has been well characterized and it has high activity in hydrolyzing the terminal a -Gal residues from glycoconjugates on human blood group B erythrocytes to demonstrate group 0 serotype. a -Gal from coffee bean has been successf u 11 y used i n the serologicl conversion of red blood cells in clinical studies of blood transfusion. The molecular weight of different sources of a-Gal is between 28 000-370 000, and the molecular weight of a-Gal from coffee bean(Coffea arabicd) is 41 000. The whole Tenth cDNA of a -Gal from coffee bean(C. arabicd) is 1384bp and that of the mat peptides cDNA is 1089bp.
The results of this study are as follows:
l.A Simple, fast, and efficiency method for extracting of total RNA from coffee bean has been used by RNA extracting Kit which were improved in this paper.
2. Two mat_peptides cDNA encoding n -Gal have been cloned by RT-PCR from coffee bean ( C. liberica & C. canephora ) . The mat_peptidcs cDNA are 1089bp, encoded 364 amino acid(AA). The cDNA from C. liberica is 98.7% homologous to the published C. arabica sequence. It has been found that 14bp of DNA sequence or 8 AA of protein sequence were changed. The cDNA of u -Gal gene from C. canephora is 99.27% homologous to the published C. arabica sequence,while 8bp of DNA sequence or 4 AA of protein sequence were changed.
3. Four expression vectors, pPICZ a A/Gal-Z, pPICZ a A/Ga1-D, pGAPZ a A/Gal-Z, pGAPZ a A/Gal-D have been constructed successfully with the a-Gal gene from coffee bean and the vectors, pPICZ a A and pGAPZ a of Pichia pasloris.
4. One Excherichia coli expression vector, pFT-22/Gal-D has been constructed with the a -Gal gene isolated from C. liberica and the E coli secretion vector pET-22b ( + ) .
5. The three constructed vectors pPICZ a A/Gal-Z, pPICZa A/Gal-D, pGAPZ a A/Gal-D were transferred into P. pastoris stain GS115. The shake-flask expression with recombinant strain of P. pastoris, pPICZ a A/Gal-Z/GS115, pPICZ a A/Gal-D/GS115, pGAPZ a A/Gal-D/GSl15; the fermentation expression with recombinant strain of P. pastoris , pPICZ a A/Gal-D/GSl15 have been made. The products of shake-flask and fermentation expression showed a main band of 40KD on SDS-PAGE.
6. The enzyme assay showed the high enzyme activity of r a -galactosidase when using pPICZ a A vector.The enzyme activity from C. liberica was 19.11 (U/ml)at 48 h and up to 48.22( U/ml )at 108 h. the en/yme activity from C. cancphora was 15.93 (U/ml) at 48 h and up to 18.18 (U/ml) at 108 h.
7. There were some differences between the r a -Gal quantities of expression with different vectors, the methanol inducing vector, pPICZ a A was better than that of the constitute vector pGAPZ a A.
8. The constructed vector pPICZa A/Gal-D has been transferred into P. pastoris stain GS115, followed by fermentation, a single protein band of 40 KD showed on SDS-PAGE. The enzyme assay showed the activity of a -galactosidase.
9. The constructed vector pET-22/Gal-D was transferred into E. coli stain DL21(DE3)pLysS, followed by shake-flask expression. There was not obviously objective protein band has been seen on SDS-PAGE. The enzyme assay showed a light activity of a -Gal. The result showed that the a -Gal gene was not well expression in this expression system.
引文
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