一种工程化核糖激酶、其编码基因、表达载体、工程菌及其应用
阅读说明：本技术 一种工程化核糖激酶、其编码基因、表达载体、工程菌及其应用 (Engineered ribokinase, coding gene, expression vector, engineering bacterium and application thereof ) 是由 李桂东 王剑峰 于 2021-04-30 设计创作，主要内容包括：本发明公开了一种分子改造的工程化核糖激酶、其编码基因、表达载体、工程菌及其应用。采用理性设计的定向进化技术筛选到一株最优突变株(G42F)。其制备方法为以pet28a为载体,利用定点突变技术将42位的甘氨酸突变成苯丙氨酸,并在大肠杆菌中培养表达。与野生型核糖激酶相比,本发明提供的工程化核糖激酶具有更高的反应活性。(The invention discloses a molecular modified engineered ribokinase, a coding gene, an expression vector, an engineering bacterium and application thereof. An optimal mutant strain is screened by a rational directed evolution technology (G42F). The preparation method comprises the steps of using pet28a as a vector, mutating glycine at position 42 into phenylalanine by using a site-directed mutagenesis technology, and culturing and expressing in escherichia coli. Compared with wild type ribokinase, the engineered ribokinase provided by the invention has higher reactivity.)
技术领域
本发明属于生物制药与生物转化领域，具体涉及一种工程化核糖激酶及其应用。
背景技术
烟酰胺单核苷酸(NMN)参与人体细胞内重要辅酶烟酰胺腺嘌呤二核苷酸(NAD)的合成，在细胞能量生成中扮演重要角色。近年来，NMN因其在抗衰老及年龄相关退行性病变方面的应用受到广泛关注，其制备方法也成为各大医药公司的研究热点。从催化剂的角度，NMN的合成主要分为化学催化和生物催化，其中生物催化具有绿色环保、高效、副产物少等特点占据优势地位。
多家公司也报道了生物催化合成NMN的方法。例如，尚科生物(CN108949865A)公开了一种固定化细胞催化5-磷酸核糖和烟酰胺反应生成NMN的方法，固定化细胞可重复利用，反应浓度可达到13.3g/L，但是，5-磷酸核糖价格较高。邦泰生物(US2018162895A1)公开了一种核糖激酶、焦磷酸合成酶、烟酰胺磷酸核糖转移酶三酶串联一锅法合成NMN，并对其中的烟酰胺磷酸核糖转移酶进行分子改造，得到了较好的反应收率，而对该串联反应第一步(核糖激酶催化生成5-磷酸核糖)没有进一步的研究。
因此，无论是分步法还是一锅法，对于改进核糖激酶催化活性的研究具有重要意义。
发明内容
本发明的目的是克服现有技术的不足，提供一种经过分子改造的工程化核苷激酶(Rbk)，还提供了工程化核糖激酶的基因，含有该基因的表达载体，工程菌株及其制备方法，以及使用该工程化核糖激酶制备5-磷酸核糖的反应工艺。
一种工程化核糖激酶，所述工程化核糖激酶的氨基酸序列如SEQ ID NO.2所示。本发明人的实验对象是一个来源于Escherichia coli的野生型核糖激酶，其氨基酸序列如序列1所示。但野生型的催化活性并不理想。发明人通过定向进化技术对该核糖激酶进行改造，得到一种工程化核糖激酶，意外地发现将42位的甘氨酸突变成苯丙氨酸后，突变株G42F具有更好的催化活性，其氨基酸序列如序列2所示。
本发明通过理性设计结合定向进化的方法对核糖激酶进行分子改造，改良后的酶在催化活性上得到提升。首先，本发明通过分析核糖激酶的晶体结构，在活性中心附近筛选出多个关键氨基酸，并通过引物设计和PCR扩增序列，分别将关键氨基酸突变成体积不同的丙氨酸、亮氨酸和苯丙氨酸；接着，用基因工程技术将序列与pET28a质粒连接；最后转化构建工程菌，表达核糖激酶。
所述的工程化核糖激酶的制备方法，包括以下步骤：
A核糖激酶基因序列引物设计、PCR扩增；
B核糖激酶基因片段与克隆载体连接，得到携带核糖激酶基因的重组表达载体；
C重组表达载体转化到宿主细菌中，得到重组体细菌；
D重组体细菌培养、提取得到含有核糖激酶的粗酶液
本发明还是提供了一种编码基因，核苷酸序列与所述的工程化核糖激酶的氨基酸序列具有对应关系。
本发明还提供了一种重组表达载体，包含载体pet28a和所述的编码基因。
本发明还提供了一种基因工程菌，包括宿主细胞和转入宿主细胞的目的基因，所述目的基因包含所述的(R,S)-羰基还原酶的编码基因。
所述的表达载体适合在大肠杆菌中表达，因此，所述宿主细胞优选为大肠杆菌。
本发明还进一步提供了一种5-磷酸核糖的制备方法，包括：
在所述的工程化核糖激酶的催化下，核糖和ATP进行反应得到5-磷酸核糖。其工艺路线如下：
作为优选，反应的温度为25～35℃，反应时间为10～15小时。
作为优选，反应溶剂为乙腈。
同现有技术相比，本发明的有益效果体现在：
本发明通过在野生型核糖激酶进行改造，经过大量的筛选，发现将42位的甘氨酸突变成苯丙氨酸后，突变株G42F具有更好的催化活性，提高催化反应的收率。
附图说明
图1为本发明的质粒连接模式示意图。
具体实施方式
下面结合具体实施例和附图进一步阐述本发明。以下实施例是对本发明的解释，但本发明并不局限于以下实施例。
实施例1：基因克隆和表达载体的构建
源自于Escherichia coli的野生型核糖激酶的氨基酸序列，可从NCBI上检索得到，如序列1所示，然后通过本领域的常见技术合成出对应的核酸并克隆到表达载体pet28a上。将重组表达质粒转化到E.coil BL21(DE3)的感受态细胞中，转化条件42℃，热击90秒，转化液涂布到含有氯霉素的LB平板上，37℃倒置培养过夜，即获得重组转化体。
实施例2：核糖激酶的表达和酶液的制备
取冻存的RK菌液5μL转入5mL(含50μL/ml硫酸卡那霉素)LB培养基中，过夜活化。
活化后的菌液接入500mL LB培养基中，37℃条件下培养至OD600约0.7左右，预冷至20℃。
加入终浓度为0.2mM的IPTG诱导剂，随后放入20℃摇床中培养20h。
将培养好的菌液离心，并用pH 8.0，50mM Tris-His缓冲液(含100mM浓度的NaCl)洗涤三次
将洗涤好的菌用pH 8.0，50mM Tris-His缓冲液(含100mM浓度的NaCl)重新悬浮，超声裂解。取出裂解后的破碎菌液离心，取上清，用于后续反应。
实施例3：核糖激酶突变文库的建立
根据核糖激酶的晶体结构，选取活性中心周围的关键氨基酸(14，16，42，46，143，265，273)，分别突变成典型代表小-中-大体积的丙氨酸(A)、亮氨酸(L)、苯丙氨酸(F)。具体方法如下：
PCR体系为：10xBuffer 5μL，2mM dNTPs5μL，质粒DNA模板1μL(50ng/μL)，上下游引物各取2μL(10μM)，高保真酶0.5μL，DMSO 1μL，ddH2O 34μL。PCR引物在突变位置的密码子如下：
突变株
引物序列
N14A
CTGGGTAGCGTTGCGGCAGATCATGTTC
N14L
CTGGGTAGCGTTCTGTGCAGATCATGTTC
N14F
CTGGGTAGCGTTTTTGCAGATCATGTTC
D16A
GTTAATGCAGCGCATGTTCTG
D16L
GTTAATGCACTGCATGTTCTG
D16F
GTTAATGCATTTCATGTTCTG
G42A
GTTATTCCGGGTGCGAAAGGCGCAAATC
G42L
GTTATTCCGGGTCTGAAAGGCGCAAATC
G42F
GTTATTCCGGGTTTTAAAGGCGCAAATC
K43A
ATTCCGGGTGGTGCGGGCGCAAATCAGGC
K43L
ATTCCGGGTGGTCTGGGCGCAAATCAGGC
K43F
ATTCCGGGTGGTTTTGGCGCAAATCAGGC
E143A
CTGATGCAGCTGGCGACCCCGCTGGATGG
E143L
CTGATGCAGCTGCTGACCCCGCTGGATGG
E143F
CTGATGCAGCTGTTTACCCCGCTGGATGG
D255A
CTGCAGCCGGTGCGACCTTTAATGGTG
D255L
CTGCAGCCGGTCTGACCTTTAATGGTG
D255F
CTGCAGCCGGTTTTACCTTTAATGGTG
S273A
TGCCTCTGGAAGCGGCAATTAAATTTG
S273L
TGCCTCTGGAACTGGCAATTAAATTTG
S273F
TGCCTCTGGAATTTGCAATTAAATTTG
PCR扩增步骤为：(1)94℃，预变性3min；(2)98℃，变性10s；(3)64℃退火30s；(4)68℃延伸4min；步骤(2)～(3)重复29次；(5)72℃继续延伸10min，冷却至4℃。在PCR产物中加入2μLDpnI，37℃过夜酶切消除质粒模板。酶切后的PCR产物用电击法转化E.coli BL21(DE3)感受态细胞，涂布到含硫酸卡那霉素的LB平板上即得目标残基位置的饱和突变文库。
实施例4：工程化核糖激酶催化生成5-磷酸核糖
反应条件：将含有5mg核糖的50μL乙腈加入1mL粗酶液中，30℃条件下反应12h。
突变株
产率(％)<sup>a</sup>
WT
71.6
N14A
10.0
N14L
22.8
N14F
25.2
D16A
19.8
D16L
59.2
D16F
41.7
G42A
35.1
G42L
69.9
G42F
87.3
K43A
30.5
K43L
29.6
K43F
45.9
E143A
26.3
E143L
39.4
E143F
43.7
D255A
23.9
D255L
41.7
D255F
50.4
S273A
37.6
S273L
42.1
S273F
62.5
a上述产率为HPLC产率。
由上述结果可知，最佳突变株位G42F，反应产率从野生型的71.6％提升到87.3％。
序列表
<110> 杭州灵犀健康科技有限公司
<120> 一种工程化核糖激酶、其编码基因、表达载体、工程菌及其应用
<160> 2
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Gly Gly Thr Ala Gly Thr Cys Ala Cys Ala Thr Gly Ala Ala Thr Ala
1 5 10 15
Ala Ala Cys Thr Gly Gly Thr Gly Gly Thr Gly Cys Thr Gly Gly Gly
20 25 30
Thr Ala Gly Cys Gly Thr Thr Ala Ala Thr Gly Cys Ala Gly Ala Thr
35 40 45
Cys Ala Thr Gly Thr Thr Cys Thr Gly Cys Ala Gly Gly Thr Thr Cys
50 55 60
Cys Gly Ala Gly Cys Thr Thr Thr Cys Cys Gly Cys Gly Thr Cys Cys
65 70 75 80
Gly Gly Gly Thr Gly Ala Ala Ala Cys Ala Cys Thr Gly Cys Ala Thr
85 90 95
Gly Gly Thr Cys Gly Thr Ala Ala Thr Thr Ala Thr Cys Ala Gly Gly
100 105 110
Thr Thr Ala Thr Thr Cys Cys Gly Gly Gly Thr Gly Gly Thr Ala Ala
115 120 125
Ala Gly Gly Cys Gly Cys Ala Ala Ala Thr Cys Ala Gly Gly Cys Gly
130 135 140
Gly Thr Thr Gly Cys Gly Gly Cys Ala Gly Cys Ala Cys Gly Thr Ala
145 150 155 160
Thr Gly Cys Ala Gly Gly Cys Ala Gly Ala Thr Gly Thr Thr Gly Gly
165 170 175
Thr Thr Thr Thr Ala Thr Thr Gly Cys Ala Thr Gly Thr Gly Thr Thr
180 185 190
Gly Gly Thr Gly Ala Thr Gly Ala Thr Ala Gly Thr Thr Thr Thr Gly
195 200 205
Gly Thr Ala Thr Thr Ala Ala Thr Ala Thr Cys Cys Gly Thr Gly Ala
210 215 220
Ala Ala Gly Cys Thr Thr Thr Ala Ala Ala Cys Thr Gly Gly Ala Thr
225 230 235 240
Gly Gly Thr Ala Thr Thr Ala Ala Thr Ala Cys Ala Gly Cys Ala Gly
245 250 255
Gly Thr Gly Thr Gly Ala Ala Ala Cys Thr Gly Cys Ala Gly Cys Cys
260 265 270
Gly Ala Ala Thr Thr Gly Thr Cys Cys Ala Ala Cys Cys Gly Gly Thr
275 280 285
Ala Thr Thr Gly Cys Gly Ala Thr Gly Ala Thr Thr Cys Ala Gly Gly
290 295 300
Thr Thr Ala Gly Thr Gly Ala Thr Ala Gly Cys Gly Gly Thr Gly Ala
305 310 315 320
Ala Ala Ala Thr Ala Gly Cys Ala Thr Thr Thr Gly Thr Ala Thr Thr
325 330 335
Ala Gly Cys Gly Cys Ala Gly Ala Ala Gly Cys Ala Ala Ala Thr Gly
340 345 350
Cys Thr Ala Ala Ala Cys Thr Gly Ala Cys Cys Gly Cys Ala Gly Cys
355 360 365
Ala Gly Cys Ala Ala Thr Thr Gly Ala Ala Cys Cys Gly Gly Ala Thr
370 375 380
Cys Thr Gly Gly Cys Ala Gly Cys Cys Ala Thr Thr Cys Gly Thr Gly
385 390 395 400
Ala Thr Gly Cys Ala Cys Gly Cys Thr Ala Thr Cys Thr Gly Cys Thr
405 410 415
Gly Ala Thr Gly Cys Ala Gly Cys Thr Gly Gly Ala Ala Ala Cys Cys
420 425 430
Cys Cys Gly Cys Thr Gly Gly Ala Thr Gly Gly Thr Ala Thr Cys Cys
435 440 445
Thr Gly Ala Ala Ala Gly Cys Ala Gly Cys Ala Cys Ala Gly Gly Ala
450 455 460
Ala Gly Cys Ala Ala Ala Ala Ala Cys Cys Gly Cys Ala Ala Ala Ala
465 470 475 480
Ala Cys Cys Ala Ala Thr Gly Thr Ala Ala Thr Cys Cys Thr Gly Ala
485 490 495
Ala Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Gly Cys Ala Cys Gly
500 505 510
Cys Gly Ala Ala Cys Thr Gly Cys Cys Gly Gly Ala Thr Gly Ala Ala
515 520 525
Cys Thr Gly Cys Thr Gly Ala Ala Ala Thr Gly Thr Gly Thr Ala Gly
530 535 540
Ala Thr Cys Thr Gly Ala Thr Cys Ala Cys Cys Cys Cys Gly Ala Ala
545 550 555 560
Thr Gly Ala Ala Ala Cys Cys Gly Ala Ala Gly Cys Ala Gly Ala Ala
565 570 575
Gly Thr Cys Cys Thr Gly Ala Cys Cys Gly Gly Cys Ala Thr Thr Ala
580 585 590
Cys Cys Gly Thr Cys Thr Ala Thr Gly Ala Thr Gly Ala Thr Ala Gly
595 600 605
Cys Thr Cys Thr Gly Cys Ala Cys Ala Gly Cys Ala Gly Gly Cys Ala
610 615 620
Gly Cys Cys Gly Ala Thr Gly Cys Ala Cys Thr Gly Cys Ala Thr Thr
625 630 635 640
Gly Thr Ala Ala Ala Gly Gly Thr Ala Thr Thr Gly Ala Ala Ala Thr
645 650 655
Thr Gly Thr Thr Ala Thr Cys Ala Thr Cys Ala Cys Cys Cys Thr Gly
660 665 670
Gly Gly Thr Ala Gly Cys Ala Ala Ala Gly Gly Thr Gly Thr Thr Thr
675 680 685
Gly Gly Cys Thr Gly Ala Gly Thr Cys Ala Gly Ala Ala Cys Gly Gly
690 695 700
Thr Cys Gly Cys Gly Gly Cys Cys Ala Gly Cys Gly Cys Ala Thr Thr
705 710 715 720
Cys Cys Gly Gly Gly Thr Thr Thr Thr Gly Thr Thr Gly Thr Thr Ala
725 730 735
Ala Ala Gly Cys Ala Ala Cys Ala Gly Ala Thr Ala Cys Cys Ala Cys
740 745 750
Thr Gly Cys Ala Gly Cys Cys Gly Gly Thr Gly Ala Thr Ala Cys Cys
755 760 765
Thr Thr Thr Ala Ala Thr Gly Gly Thr Gly Cys Cys Cys Thr Gly Gly
770 775 780
Thr Thr Ala Cys Thr Gly Gly Thr Cys Thr Gly Cys Thr Gly Cys Ala
785 790 795 800
Gly Gly Ala Ala Ala Thr Gly Cys Cys Thr Cys Thr Gly Gly Ala Ala
805 810 815
Ala Gly Cys Gly Cys Ala Ala Thr Thr Ala Ala Ala Thr Thr Thr Gly
820 825 830
Cys Ala Cys Ala Thr Gly Cys Ala Gly Cys Ala Gly Cys Cys Gly Cys
835 840 845
Ala Ala Thr Thr Ala Gly Cys Gly Thr Thr Ala Cys Cys Cys Gly Thr
850 855 860
Thr Thr Thr Gly Gly Cys Gly Cys Ala Cys Ala Gly Ala Cys Cys Thr
865 870 875 880
Cys Ala Ala Thr Thr Cys Cys Gly Ala Cys Gly Cys Gly Thr Gly Cys
885 890 895
Ala Gly Ala Ala Gly Thr Thr Gly Ala Ala Gly Cys Ala Thr Thr Thr
900 905 910
Cys Thr Gly Gly Cys Ala Gly Ala Ala Cys Ala Thr Thr Cys Cys Thr
915 920 925
Ala Ala
930
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<211> 930
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 2
Gly Gly Thr Ala Gly Thr Cys Ala Cys Ala Thr Gly Ala Ala Thr Ala
1 5 10 15
Ala Ala Cys Thr Gly Gly Thr Gly Gly Thr Gly Cys Thr Gly Gly Gly
20 25 30
Thr Ala Gly Cys Gly Thr Thr Ala Ala Thr Gly Cys Ala Gly Ala Thr
35 40 45
Cys Ala Thr Gly Thr Thr Cys Thr Gly Cys Ala Gly Gly Thr Thr Cys
50 55 60
Cys Gly Ala Gly Cys Thr Thr Thr Cys Cys Gly Cys Gly Thr Cys Cys
65 70 75 80
Gly Gly Gly Thr Gly Ala Ala Ala Cys Ala Cys Thr Gly Cys Ala Thr
85 90 95
Gly Gly Thr Cys Gly Thr Ala Ala Thr Thr Ala Thr Cys Ala Gly Gly
100 105 110
Thr Thr Ala Thr Thr Cys Cys Gly Gly Gly Thr Thr Thr Thr Ala Ala
115 120 125
Ala Gly Gly Cys Gly Cys Ala Ala Ala Thr Cys Ala Gly Gly Cys Gly
130 135 140
Gly Thr Thr Gly Cys Gly Gly Cys Ala Gly Cys Ala Cys Gly Thr Ala
145 150 155 160
Thr Gly Cys Ala Gly Gly Cys Ala Gly Ala Thr Gly Thr Thr Gly Gly
165 170 175
Thr Thr Thr Thr Ala Thr Thr Gly Cys Ala Thr Gly Thr Gly Thr Thr
180 185 190
Gly Gly Thr Gly Ala Thr Gly Ala Thr Ala Gly Thr Thr Thr Thr Gly
195 200 205
Gly Thr Ala Thr Thr Ala Ala Thr Ala Thr Cys Cys Gly Thr Gly Ala
210 215 220
Ala Ala Gly Cys Thr Thr Thr Ala Ala Ala Cys Thr Gly Gly Ala Thr
225 230 235 240
Gly Gly Thr Ala Thr Thr Ala Ala Thr Ala Cys Ala Gly Cys Ala Gly
245 250 255
Gly Thr Gly Thr Gly Ala Ala Ala Cys Thr Gly Cys Ala Gly Cys Cys
260 265 270
Gly Ala Ala Thr Thr Gly Thr Cys Cys Ala Ala Cys Cys Gly Gly Thr
275 280 285
Ala Thr Thr Gly Cys Gly Ala Thr Gly Ala Thr Thr Cys Ala Gly Gly
290 295 300
Thr Thr Ala Gly Thr Gly Ala Thr Ala Gly Cys Gly Gly Thr Gly Ala
305 310 315 320
Ala Ala Ala Thr Ala Gly Cys Ala Thr Thr Thr Gly Thr Ala Thr Thr
325 330 335
Ala Gly Cys Gly Cys Ala Gly Ala Ala Gly Cys Ala Ala Ala Thr Gly
340 345 350
Cys Thr Ala Ala Ala Cys Thr Gly Ala Cys Cys Gly Cys Ala Gly Cys
355 360 365
Ala Gly Cys Ala Ala Thr Thr Gly Ala Ala Cys Cys Gly Gly Ala Thr
370 375 380
Cys Thr Gly Gly Cys Ala Gly Cys Cys Ala Thr Thr Cys Gly Thr Gly
385 390 395 400
Ala Thr Gly Cys Ala Cys Gly Cys Thr Ala Thr Cys Thr Gly Cys Thr
405 410 415
Gly Ala Thr Gly Cys Ala Gly Cys Thr Gly Gly Ala Ala Ala Cys Cys
420 425 430
Cys Cys Gly Cys Thr Gly Gly Ala Thr Gly Gly Thr Ala Thr Cys Cys
435 440 445
Thr Gly Ala Ala Ala Gly Cys Ala Gly Cys Ala Cys Ala Gly Gly Ala
450 455 460
Ala Gly Cys Ala Ala Ala Ala Ala Cys Cys Gly Cys Ala Ala Ala Ala
465 470 475 480
Ala Cys Cys Ala Ala Thr Gly Thr Ala Ala Thr Cys Cys Thr Gly Ala
485 490 495
Ala Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Gly Cys Ala Cys Gly
500 505 510
Cys Gly Ala Ala Cys Thr Gly Cys Cys Gly Gly Ala Thr Gly Ala Ala
515 520 525
Cys Thr Gly Cys Thr Gly Ala Ala Ala Thr Gly Thr Gly Thr Ala Gly
530 535 540
Ala Thr Cys Thr Gly Ala Thr Cys Ala Cys Cys Cys Cys Gly Ala Ala
545 550 555 560
Thr Gly Ala Ala Ala Cys Cys Gly Ala Ala Gly Cys Ala Gly Ala Ala
565 570 575
Gly Thr Cys Cys Thr Gly Ala Cys Cys Gly Gly Cys Ala Thr Thr Ala
580 585 590
Cys Cys Gly Thr Cys Thr Ala Thr Gly Ala Thr Gly Ala Thr Ala Gly
595 600 605
Cys Thr Cys Thr Gly Cys Ala Cys Ala Gly Cys Ala Gly Gly Cys Ala
610 615 620
Gly Cys Cys Gly Ala Thr Gly Cys Ala Cys Thr Gly Cys Ala Thr Thr
625 630 635 640
Gly Thr Ala Ala Ala Gly Gly Thr Ala Thr Thr Gly Ala Ala Ala Thr
645 650 655
Thr Gly Thr Thr Ala Thr Cys Ala Thr Cys Ala Cys Cys Cys Thr Gly
660 665 670
Gly Gly Thr Ala Gly Cys Ala Ala Ala Gly Gly Thr Gly Thr Thr Thr
675 680 685
Gly Gly Cys Thr Gly Ala Gly Thr Cys Ala Gly Ala Ala Cys Gly Gly
690 695 700
Thr Cys Gly Cys Gly Gly Cys Cys Ala Gly Cys Gly Cys Ala Thr Thr
705 710 715 720
Cys Cys Gly Gly Gly Thr Thr Thr Thr Gly Thr Thr Gly Thr Thr Ala
725 730 735
Ala Ala Gly Cys Ala Ala Cys Ala Gly Ala Thr Ala Cys Cys Ala Cys
740 745 750
Thr Gly Cys Ala Gly Cys Cys Gly Gly Thr Gly Ala Thr Ala Cys Cys
755 760 765
Thr Thr Thr Ala Ala Thr Gly Gly Thr Gly Cys Cys Cys Thr Gly Gly
770 775 780
Thr Thr Ala Cys Thr Gly Gly Thr Cys Thr Gly Cys Thr Gly Cys Ala
785 790 795 800
Gly Gly Ala Ala Ala Thr Gly Cys Cys Thr Cys Thr Gly Gly Ala Ala
805 810 815
Ala Gly Cys Gly Cys Ala Ala Thr Thr Ala Ala Ala Thr Thr Thr Gly
820 825 830
Cys Ala Cys Ala Thr Gly Cys Ala Gly Cys Ala Gly Cys Cys Gly Cys
835 840 845
Ala Ala Thr Thr Ala Gly Cys Gly Thr Thr Ala Cys Cys Cys Gly Thr
850 855 860
Thr Thr Thr Gly Gly Cys Gly Cys Ala Cys Ala Gly Ala Cys Cys Thr
865 870 875 880
Cys Ala Ala Thr Thr Cys Cys Gly Ala Cys Gly Cys Gly Thr Gly Cys
885 890 895
Ala Gly Ala Ala Gly Thr Thr Gly Ala Ala Gly Cys Ala Thr Thr Thr
900 905 910
Cys Thr Gly Gly Cys Ala Gly Ala Ala Cys Ala Thr Thr Cys Cys Thr
915 920 925
Ala Ala
930
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