CN87105519A - 毕赤酵母产生的亲脂蛋白的纯化 - Google Patents
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Abstract
本发明叙述了一种在促溶盐存在的条件下进行细胞裂解,从毕赤酵母属转化细胞选择性地提取所需亲脂蛋白的方法。在本发明细胞裂解条件下,所提取的蛋白质总量减少,而所需亲脂蛋白质的回收量保持相对恒定,从而与对照细胞提取物相比较,得到了一种含有高浓度所需亲脂蛋白的细胞提取物。
Description
这项发明与蛋白质的回收与纯化有关。一方面本项发明与从细胞碎片中分离可溶性蛋白质有关,另一方面它又与蛋白质的选择性提取有关。
重组DNA技术已迅速成为生产用于诊断、治疗,化学应用等方面的肽和蛋白质的有力工具。而经常遇到的一个问题是要获得不混有其他蛋白的所需纯蛋白质,这些其他蛋白也是在表达所需蛋白质过程中产生的。本项发明是为了改进通过重组DNA技术生产的蛋白质的回收方法。
此项发明的一个目的就是提供一种有效地回收由遗传修饰的酵母产生的所需蛋白质的方法。
通过审查本文提供的公开内容和权利要求可以明确本发明的这种和它种目的。
按照本发明,可以发现遗传修饰的毕赤酵母菌株产生的亲脂蛋白可以采用一种含促溶盐的裂解缓冲液选择性地回收,在这种裂解缓冲液存在的条件下,破裂毕赤酵母会减少所抽提蛋白质的总量,同时对所要的亲脂蛋白质的提取没有影响,这样实施本发明所得到的可溶性细胞提取物含有较抽提物中所有其它蛋白质含量高的亲脂蛋白,因此简化了所需亲脂蛋白质的进一步纯化步骤。
按照本发明,毕赤酵母属宿主细胞产生的亲脂蛋白质由下法提取:
(a)在细胞破裂条件下处理细胞足够长时间,使所有细胞完全破裂;细胞破裂是在存在下列组分的提取介质中进行的;
至少一种促溶盐,浓度范围为1M-8M
介质的缓冲PH值要适于保持所需蛋白稳定,一般PH在6~8范围。
b.回收从步骤(a)得到的可溶部分。为了进一步浓缩和提纯所需蛋白,根据本发明,回收的可溶性部分可以采用本领域技术人员所熟知的技术方法进行进一步处理。这样可溶部分中的蛋白质可以采用透析,通过反相树脂后用最小体积溶剂洗脱、沉淀、超滤,冷冻干燥等方法浓缩。适用于进一步纯化目的蛋白的技术有使用筛析树脂进行分级分离,高效液相层析,离子交换,疏水层析等。
在本说明书中所用的术语“亲脂蛋白质”是指那些倾向于与脂质膜结合的蛋白,或在脂或类脂物质存在下,聚合形成类似微团结构的蛋白。典型的这类蛋白质一般有高含量的疏水氨基酸,如,异亮氨酸、缬氨酸、亮氨酸、苯丙氨酸、色氨酸和丙氨酸。
典型的亲脂蛋白包括,但不限于:
所有形式的乙型肝炎表面抗原,包括S形,前S1形,前S2形等;
磷脂酰丝氨酸脱羧酶(自大肠杆菌);
λ噬菌体D蛋白;
低密度脂蛋白(LDL);
高密度脂蛋白(HDL);以及
二氢乳清酸脱氢酶。
在本说明书中所用的术语“促溶盐”是指那些其阴离子有利于无极性基团向水转移的盐类。这类盐包括含硫氰酸盐离子,卤素离子如碘化物和溴化物,低卤素离子如过氯酸盐,以及阳离子如锂、钙、钡的化合物。
本发明实施中采用的典型促溶盐类包括硫氰酸钠、硫氰酸钾、碘化钠、碘化钾、次氯酸钠、氯化锂、溴化锂、盐酸胍、硫氰酸胍、尿素等。
采用经过遗传修饰,带有编码所需蛋白DNA序列的毕赤酵母作合适宿主菌,可以使毕赤酵母产生亲脂蛋白。本领域技术人员能够采用各种方法很容易地获得编码所需亲脂蛋白的合适DNA序列,如从天然来源中分离,构建合成DNA序列等。毕赤酵母菌中DNA操作的特殊技术见于《分子和细胞生物学》第五卷,第1111页与第3376页(1985)的文章中。
在足以使所有细胞破碎的条件下,按发明步骤对细胞进行提取。细胞破裂是在一种抽提介质中进行的,这种介质含至少一种促溶盐,浓度大约在1M-8M范围,此促溶盐在合适pH(一般是6-8)的中介缓冲液中,以维持目的蛋白处于稳定状态。为使抽提过程中蛋白质尽少降解,可以将抗蛋白酶制剂,如苯甲磺酰氟加进裂解缓冲液中。
在实施本发明中,细胞破裂一般要用一种有凸缘的研磨器(bead mill)或类似装置进行均浆。细胞破裂所需要的时间取决于细胞壁对破裂敏感度,均化条件的剧烈程度,裂解缓冲液中所加的化合物及其浓度等。一般是在破碎条件下处理细胞0.5至30分钟;最好是1至5分钟。
细胞破裂的温度要大体上控制以使降解蛋白质的酶的作用减到最小。这样,细胞破裂一般在0到10℃的温度范围内进行,0℃更可取,以使破裂过程中目的蛋白质的降解减到最小,因此就能增加从破裂细胞中回收的目的蛋白的量。
一旦细胞破裂,可通过利用本领域技术人员熟知的技术如离心或切向过滤来去除细胞碎片以回收可溶部分,所得到的无细胞肉汤较仅破裂细胞然后从其中回收可溶部分所得的肉汤有更多的所需亲脂蛋白质。
如果需要,这样处理过的肉汤可以采用此领域技术人员熟知的方法,如酸沉淀、过滤、层析、溶剂蒸发等作进一步处理以回收所需亲脂蛋白的浓缩部分。
参考以下非限定例子,下面将更详尽地介绍本发明。
例一
下面叙述由载体pBSAG151(从美国农业部北方地区研究中心,Peoria ILL,大肠杆菌宿主中获得,编号为NNRLB-18021)转化的毕赤酵母中提取22nm乙肝表面抗原颗粒。
培养巴斯德毕赤酵母,生长直至在660nm,每毫升培养液光密度值达到10-100。将光密度单位相当于100的菌液部分转移到13×100毫米硼硅酸盐培养管中,并用20倍体积的裂解缓冲液(配方如下)冲洗两遍。
在沉淀了的细胞(IEC临床离心机)中加入0.5克酸洗过的玻璃珠(0.5毫米),然后加0.35毫升裂解缓冲液。裂解缓冲液中含有0.5M Nacl及0.1%Triton×-100(重量/体积),(作为对照物),也可代之以2M或3M促溶盐(0.1%Triton×-100含否皆可)。所有溶液都用10mM磷酸钠在PH7.5缓冲处理。用混合器搅拌混合液8分钟,每隔1分钟,以最大速度搅拌一次。间歇之间混合液要在冰上冷却不少于一分钟。培养试管最好保持20-40度倾斜,这样振荡能达到最大程度破碎细胞。完成裂解后,除去破裂细胞的溶液,用0.35毫升裂解缓冲液冲洗玻璃珠;然后合并这两种溶液并在13,000g速度下离心15分钟,除去上清液并检测乙型肝炎病毒表面抗原颗粒的免疫活性(Ausria检测)及总蛋白含量(Bradford)。结果见表一(a)。
例二
为进一步说明本发明的方法,将80毫升悬浮液(1份细胞团:2份裂解缓冲液,体积/体积)用64毫米搅拌器以4500rpm的速度进行细胞破碎。裂解缓冲液含有0.5M Nacl和0.1%Triton(重量/体积比)或含3M硫氰酸钾。测定上清液乙肝病毒表面抗原(Ausria)及总蛋白(Bradford法)。结果见表一(b)。
没有一种促溶盐(碘化钾或硫氰化钾)存在的条件下,产生明显高于对照的乙型肝炎病毒表面抗原颗粒值(栏Ⅰ);这说明促溶盐类抑制了总蛋白质的释放(栏Ⅱ),从而增加了乙肝表面抗原颗粒的特异性活性2-5倍(栏Ⅲ)。
这些例子仅仅是为了说明本发明的应用,而不应该理解为对本发明的范围或所附的权利要求以任何方式进行限制。不违背发明精神和实质的合理改变和修正,被认为属于所需和寻求的专利保护之列。
Claims (9)
1、一种从毕赤酵母属宿主细胞中提取亲脂性蛋白质的方法,它包括:
(a)在足以使所有细胞破裂的时间条件下破裂细胞,其中所说的细胞破裂是在由下列成分组成的一种提取介质存在的条件下进行的:
浓度范围为1M至8M的至少一种促溶盐,它存在于一种经缓冲过的其pH值适合于维持所说亲脂蛋白质稳定的介质中;
(b)回收从步骤(a)所得到的可溶性部分。
2、如权利要求1所述的方法,其中所说的PH维持在6至8的范围内。
3、如权利要求1所述的方法,其中所说的亲脂蛋白质是一种倾向于和脂膜结合的蛋白质。
4、如权利要求1所述的方法,其中所说的亲脂蛋白质是一种在脂或类脂物质存在的情况下聚集成类似分子团结构的蛋白质。
5、如权利要求1所述的方法,其中所说的亲脂蛋白质选自:
S型乙型肝炎表面抗原,
前S1型乙型肝炎表面抗原,
前S2型乙型肝炎表面抗原,
磷脂酰丝氨酸脱羧酶(来自大肠杆菌),
λ.噬菌体D-蛋白,
低密度脂蛋白(LDL),
高密度脂蛋白(HDL),以及
二氢乳氢酸脱氢酶。
6、如权利要求1所述的方法,其中所说的破裂是在温度为0-10℃、时间0.5-30分钟的条件下进行。
7、如权利要求1所述的方法,其中所说的促溶盐选自:
硫氰酸钠,
硫氰酸钾,
碘化钠,
碘化钾,
次氯酸钠,
氯化锂,
溴化锂,
盐酸胍
硫氰酸胍
尿素
促溶盐也可以是上述两种或多种物质的混合物。
8、如权利要求1所述的方法,其中步骤(b)所说的可溶性部分的回收是通过对含有破碎细胞的溶液进行离心而完成的。
9、如权利要求1所述的方法,它进一步包括:
(c)处理从步骤(b)所得到的可溶性部分,以获得亲脂性蛋白质的浓缩物。
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US06/920,385 US4683293A (en) | 1986-10-20 | 1986-10-20 | Purification of pichia produced lipophilic proteins |
US920385 | 2001-08-01 |
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CN87105519A true CN87105519A (zh) | 1988-05-04 |
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-
1986
- 1986-10-20 US US06/920,385 patent/US4683293A/en not_active Expired - Lifetime
-
1987
- 1987-05-28 CA CA000538267A patent/CA1277272C/en not_active Expired - Lifetime
- 1987-07-31 NZ NZ221296A patent/NZ221296A/xx unknown
- 1987-07-31 PH PH35608A patent/PH23461A/en unknown
- 1987-07-31 ZA ZA875698A patent/ZA875698B/xx unknown
- 1987-08-02 IL IL83402A patent/IL83402A0/xx unknown
- 1987-08-10 IN IN621/CAL/87A patent/IN166069B/en unknown
- 1987-08-11 CN CN87105519A patent/CN1006808B/zh not_active Expired
- 1987-08-18 AU AU77178/87A patent/AU586758B2/en not_active Ceased
- 1987-09-02 KR KR870009721A patent/KR880005259A/ko not_active Application Discontinuation
- 1987-09-03 JP JP62219311A patent/JPS63105693A/ja active Pending
- 1987-10-15 PT PT85935A patent/PT85935B/pt not_active IP Right Cessation
- 1987-10-16 YU YU191387A patent/YU46080B/sh unknown
- 1987-10-16 HU HU874664A patent/HU202554B/hu not_active IP Right Cessation
- 1987-10-19 PL PL1987268298A patent/PL154027B1/pl unknown
- 1987-10-19 DK DK545187A patent/DK545187A/da not_active Application Discontinuation
- 1987-10-19 IE IE872807A patent/IE872807L/xx unknown
- 1987-10-19 FI FI874597A patent/FI874597A/fi not_active Application Discontinuation
- 1987-10-19 NO NO874357A patent/NO874357L/no unknown
- 1987-10-19 CS CS877529A patent/CS752987A2/cs unknown
- 1987-10-19 EP EP87115255A patent/EP0271667A1/en not_active Withdrawn
- 1987-10-19 DD DD87308079A patent/DD262673A5/de not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
KR880005259A (ko) | 1988-06-28 |
DK545187A (da) | 1988-04-21 |
JPS63105693A (ja) | 1988-05-10 |
CA1277272C (en) | 1990-12-04 |
CN1006808B (zh) | 1990-02-14 |
DD262673A5 (de) | 1988-12-07 |
PT85935B (pt) | 1990-07-31 |
NO874357L (no) | 1988-04-21 |
ZA875698B (en) | 1988-04-27 |
FI874597A0 (fi) | 1987-10-19 |
HUT45079A (en) | 1988-05-30 |
PL268298A1 (en) | 1988-07-07 |
IE872807L (en) | 1988-04-20 |
FI874597A (fi) | 1988-04-21 |
YU46080B (sh) | 1992-12-21 |
PT85935A (en) | 1987-11-01 |
AU586758B2 (en) | 1989-07-20 |
NO874357D0 (no) | 1987-10-19 |
YU191387A (en) | 1989-06-30 |
IN166069B (zh) | 1990-03-10 |
PH23461A (en) | 1989-08-07 |
CS752987A2 (en) | 1991-08-13 |
US4683293A (en) | 1987-07-28 |
EP0271667A1 (en) | 1988-06-22 |
IL83402A0 (en) | 1988-01-31 |
NZ221296A (en) | 1989-07-27 |
AU7717887A (en) | 1988-04-21 |
HU202554B (en) | 1991-03-28 |
PL154027B1 (en) | 1991-06-28 |
DK545187D0 (da) | 1987-10-19 |
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