实施例:
本实施例提供一种亲和兼容性链霉亲和素突变体及其制备方法。
1.全人工基因合成和突变引入
1.1已知的具有127个氨基酸的链霉亲和素成熟氨基酸,其氨基酸序列如序列表中SEQ ID NO:3所示,在其N端添加Met氨基酸作为起始密码安置,形成128个氨基酸序列,并在其中引入两个氨基酸的定点突变:S52G、R53D,以及两个氨基酸的换位突变:Y22N、N23Y,得到的128个氨基酸的突变序列(SEQ ID NO:9);
1.2将上述128个氨基酸的突变序列经美国国家生化信息中心(NCBI)网上软件DNAWorks逆翻译成核苷酸序列,上述核苷酸序列的密码子经DNAWorks优化(E. coli class II),并且在其5’-端添加了NcoI酶切位点,3’-端添加终止密码TAA和XhoI酶切位点,在上下游酶切位点外侧各加4个保护碱基,DNA合成仪合成10条寡核苷酸引物(SEQ ID NO:11~20);
1.3将上述10条寡核苷酸引物通过PCR合成人工DNA序列为403个碱基对的脱氧核糖核酸,所述人工DNA序列编码128个氨基酸的突变序列,命名为SA128m4(SEQ ID NO:21),包含4个氨基酸的突变,分别为Y22N、N23Y、S52G、R53D(SEQ ID NO:9);
1.4将PCR产物克隆进盘古基因科技(苏州)有限公司的TOPO克隆载体TopFastTM pACK4a-Bs,挑取单克隆扩增、PCR鉴定重组体;
1.5通过DNA序列分析获得了序列正确的SA128m4克隆(命名为pACK4a-SA128m4)。
2.亚克隆技术构建原核表达载体
2.1用NcoI/XhoI双酶切消化pACK4a-SA128m4,琼脂糖电泳和胶回收法回收SA128m4目的片段,同法用NcoI/XhoI处理pET28a载体,胶回收纯化;
2.2 T4 DNA连接酶连接,转化Top10感受态菌,挑取单菌落鉴定阳性克隆;
2.3测序分析验证插入片段正确,取得pET28a-SA128m4重组表达载体(如图1所示);
2.4用该载体转化BL21(DE3)感受态菌,取得表达菌株BL21-pET28a-SA128m4。
3.测试表达
3.1LB培养基扩增表达菌株BL21-pET28a-SA128m4,IPTG诱导表达,诱导条件:IPTG 0.1mMole/L,25℃诱导4小时;
3.2取1mL菌液,离心收集菌体,PBS洗涤1次,0.5mL PBS重悬浮,超声裂解菌体;
3.3在4℃,12,000RPM,离心15min,保留上清和沉淀,分别上样SDA-PAGE,考马斯亮蓝染色-脱色,观察表达条带。SA128m4的表达量占细菌总蛋白的8%,其中可溶性表达>90%。
4.SA128m4蛋白质纯化
4.1 LB+M9培养基2L,摇瓶扩菌至OD≈0.5;
4.2加IPTG诱导(0.1mMole/L), 25℃诱导表达4小时;
4.3收集菌体,超声破碎菌体,离心收集上清;
4.4饱和硫酸铵(加至80%)共沉淀,离心收集沉淀,去离子水重悬;
4.5分子筛Sephadex G-50,分选加脱盐,5%甘油水溶液洗柱,收集SA128m4蛋白组份,20mM 磷酸盐缓冲液,pH5.0(含10mM NaCl, 5%甘油)透析平衡;
4.6Mono S 阳离子交换层析:20mM磷酸盐缓冲液,pH5.0(含10mM NaCl,5% Glycerol)平衡离子交换柱,上样,5倍柱体积平衡液淋洗,20mM磷酸盐缓冲液,pH7.0(含100mM NaCl, 5% Glycerol),洗脱目标产物SA128m4;
4.7Mono Q 阴离子交换层析: pH8.5,20mM 的Tris-Cl缓冲液(含5% Glycerol)平衡色谱柱,将上一步收集的SA组份以柱平衡液透析平衡后上柱,5倍柱体积淋洗,离子梯度洗脱SA(20mM 的磷酸盐缓冲液,pH8.5,200mM NaCl);
4.8Sephadex G-200 凝胶过滤层析( 60cm×5cm),收集合并含有SA的活性峰,并对其含量,纯度进行鉴定,超滤浓缩。
5.SA128m4活性测定
BioCORE 100生物传感器测定亲和力,SA128m4与Biotin的Kd = 1.52×10-8M/L,与2-亚氨基生物素的Kd = 1.23×10-6M/L。
SA128m4的比活性(Specific Activity)达到14.8U/mg (wtSA的比活性为13~16U/mg,1U结合Biotin 1μg)。
SA128m4与生物素结合的半寿期为12小时(据报道,wtSA为6.6小时,单体SA(Monomeric SA)为3分钟)。
本发明尚有多种实施方式,凡采用等同变换或者等效变换而形成的所有技术方案,均落在本发明的保护范围之内。
<110>盘古基因科技(苏州)有限公司
<120>一种亲和兼容性链霉亲和素突变体及其制备方法
<160>21
<210>1
<211>183
<212>PRT
<213>链霉亲和素
<400>1
Met Arg Lys Ile Val Val Ala Ala Ile Ala Val Ser Leu Thr Thr
1 5 10 15
Val Ser Ile Thr Ala Ser Ala Ser Ala Asp Pro Ser Lys Asp Ser
20 25 30
Lys Ala Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp
35 40 45
Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp
50 55 60
Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu
65 70 75
Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr
80 85 90
Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys Asn
95 100 105
Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr
110 115 120
Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr
125 130 135
Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly
140 145 150
His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp
155 160 165
Ala Ala Lys Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala
170 175 180
Val Gln Gln
<210>2
<211>159
<212>PRT
<213>链霉亲和素159个成熟氨基酸
<400>2
Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala
1 5 10 15
Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile
20 25 30
Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser
35 40 45
Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr
50 55 60
Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp
65 70 75
Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr
80 85 90
Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn
95 100 105
Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp
110 115 120
Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro
125 130 135
Ser Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn Asn
140 145 150
Gly Asn Pro Leu Asp Ala Val Gln Gln
155
<210>3
<211>127
<212>PRT
<213>链霉亲和素127个核心氨基酸
<400>3
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser
1 5 10 15
Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr
20 25 30
Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr
35 40 45
Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
50 55 60
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His
65 70 75
Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala
80 85 90
Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
95 100 105
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys
110 115 120
Val Lys Pro Ser Ala Ala Ser
125
<210>4
<211>127
<212>PRT
<213>链霉亲和素127个核心氨基酸突变体S52G/R53D
<400>4
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser
1 5 10 15
Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr
20 25 30
Tyr Glu Ser Ala Val Gly Asn Ala Glu Gly Asp Tyr Val Leu Thr
35 40 45
Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
50 55 60
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His
65 70 75
Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala
80 85 90
Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
95 100 105
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys
110 115 120
Val Lys Pro Ser Ala Ala Ser
125
<210>5
<211>127
<212>PRT
<213>链霉亲和素127个核心氨基酸突变体S52G/R53N
<400>5
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser
1 5 10 15
Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr
20 25 30
Tyr Glu Ser Ala Val Gly Asn Ala Glu Gly Asn Tyr Val Leu Thr
35 40 45
Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
50 55 60
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His
65 70 75
Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala
80 85 90
Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
95 100 105
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys
110 115 120
Val Lys Pro Ser Ala Ala Ser
125
<210>6
<211>127
<212>PRT
<213>链霉亲和素127个核心氨基酸突变体S52G/R53S
<400>6
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser
1 5 10 15
Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr
20 25 30
Tyr Glu Ser Ala Val Gly Asn Ala Glu Gly Ser Tyr Val Leu Thr
35 40 45
Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
50 55 60
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His
65 70 75
Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala
80 85 90
Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
95 100 105
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys
110 115 120
Val Lys Pro Ser Ala Ala Ser
125
<210>7
<211>127
<212>PRT
<213>链霉亲和素127个核心氨基酸突变体N23A/S27E
<400>7
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Ala Gln Leu Gly Glu
1 5 10 15
Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr
20 25 30
Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr
35 40 45
Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
50 55 60
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His
65 70 75
Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala
80 85 90
Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
95 100 105
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys
110 115 120
Val Lys Pro Ser Ala Ala Ser
125
<210>8
<211>127
<212>PRT
<213>链霉亲和素127个核心氨基酸突变体N23A/S27D
<400>8
Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Ala Gln Leu Gly Asp
1 5 10 15
Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr
20 25 30
Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr
35 40 45
Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala
50 55 60
Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His
65 70 75
Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala
80 85 90
Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
95 100 105
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys
110 115 120
Val Lys Pro Ser Ala Ala Ser
125
<210>9
<211>128
<212>PRT
<213>链霉亲和素突变体SA128m4
<400>9
Met Ala Glu Ala Gly Ile Thr Gly Thr Trp Asn Tyr Gln Leu Gly
1 5 10 15
Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly
20 25 30
Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Gly Asp Tyr Val Leu
35 40 45
Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr
50 55 60
Ala Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala
65 70 75
His Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu
80 85 90
Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu
95 100 105
Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr
110 115 120
Lys Val Lys Pro Ser Ala Ala Ser
125
<210>10
<211>384
<212>DNA
<213>链霉亲和素突变体SA128m4
<400>10
atggcggaag cgggtatcac gggcacctgg aactaccagc tcggttctac cttcatcgtt 60
accgcgggtg cggacggtgc gctgaccggt acttacgaat ctgcggttgg taatgcggaa 120
ggtgactacg ttctgacggg tcgttacgac agcgcgcctg caaccgacgg ttctggtacg 180
gcgctgggtt ggaccgttgc gtggaagaac aactaccgta acgcgcactc tgcgaccacc 240
tggtctggtc agtacgttgg tggcgctgaa gcgcgtatca acacccagtg gctgctgacc 300
tctggcacca ccgaagcgaa cgcatggaaa tctaccctgg ttggtcacga caccttcacc 360
aaagttaaac cgtctgcggc gtct 384
<210>11
<211>46
<212>DNA
<213>人工合成
<400>11
aaaaccatgg cggaagcggg tatcacgggc acctggaact accagc 46
<210>12
<211>60
<212>DNA
<213>人工合成
<400>12
gcgcaccgtc cgcacccgcg gtaacgatga aggtagaacc gagctggtag ttccaggtgc 60
<210>13
<211>60
<212>DNA
<213>人工合成
<400>13
tgcggacggt gcgctgaccg gtacttacga atctgcggtt ggtaatgcgg aaggtgacta 60
<210>14
<211>60
<212>DNA
<213>人工合成
<400>14
gtcggttgca ggcgcgctgt cgtaacgacc cgtcagaacg tagtcacctt ccgcattacc 60
<210>15
<211>60
<212>DNA
<213>人工合成
<400>15
gcgcctgcaa ccgacggttc tggtacggcg ctgggttgga ccgttgcgtg gaagaacaac 60
<210>16
<211>60
<212>DNA
<213>人工合成
<400>16
tactgaccag accaggtggt cgcagagtgc gcgttacggt agttgttctt ccacgcaacg 60
<210>17
<211>60
<212>DNA
<213>人工合成
<400>17
ccacctggtc tggtcagtac gttggtggcg ctgaagcgcg tatcaacacc cagtggctgc 60
<210>18
<211>60
<212>DNA
<213>人工合成
<400>18
agggtagatt tccatgcgtt cgcttcggtg gtgccagagg tcagcagcca ctgggtgttg 60
<210>19
<211>60
<212>DNA
<213>人工合成
<400>19
gaacgcatgg aaatctaccc tggttggtca cgacaccttc accaaagtta aaccgtctgc 60
<210>20
<211>41
<212>DNA
<213>人工合成
<400>20
aaaactcgag ttaagacgcc gcagacggtt taactttggt g 41
<210>21
<211>403
<212>DNA
<213>人工合成
<400>21
aaaaccatgg cggaagcggg tatcacgggc acctggaact accagctcgg ttctaccttc 60
atcgttaccg cgggtgcgga cggtgcgctg accggtactt acgaatctgc ggttggtaat 120
gcggaaggtg actacgttct gacgggtcgt tacgacagcg cgcctgcaac cgacggttct 180
ggtacggcgc tgggttggac cgttgcgtgg aagaacaact accgtaacgc gcactctgcg 240
accacctggt ctggtcagta cgttggtggc gctgaagcgc gtatcaacac ccagtggctg 300
ctgacctctg gcaccaccga agcgaacgca tggaaatcta ccctggttgg tcacgacacc 360
ttcaccaaag ttaaaccgtc tgcggcgtct taactcgagt ttt 403