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【讨论帖】蛋白要怎么优化条件才能上清表达？
【讨论帖】蛋白要怎么优化条件才能上清表达？
我所要表达的蛋白是在PET-30a载体上表达的，虽然在包含体内表达量极高，但是我需要的是上清表达的蛋白，各种IPTG浓度，温度（37度，28度，16度）都试过了还是不行，后来又换了PET-28a和PGEX-6P-1两个载体还是不能表达在上清里面，求各位了解蛋白表达的大侠指点迷津、、
查看完整版本请点击这里：
【讨论帖】蛋白要怎么优化条件才能上清表达？
【讨论帖】蛋白要怎么优化条件才能上清表达？
最新回复
one (2013-10-06 15:42:11)
看是什么蛋白了。膜蛋白，多功能区域蛋白，需要大量辅基的蛋白，含有大量二硫键的蛋白，含有大量的辅氨酸的蛋白，这些都很难原核表达。
wzqzy (2013-10-06 15:42:57)
QUOTE:
相关疾病：口蹄疫
我的蛋白是一个蛋白酶，口蹄疫的3c蛋白，没有预测是不是含有二硫键，也不知道脯氨酸的含量呵呵，新手上路比较盲目，是不是做蛋白表达前是不是要考虑好多因素啊，我的是老板直接让做的，没有考虑太多，他也没说。我只知道这个蛋白的疏水氨基酸不多，老板老是说很好表达，但就是不上请表达，很无语，求指点啊
vivian4123 (2013-10-06 15:43:24)
QUOTE:
为什么一定要上清表达啊。不行就在哺乳动物细胞里试试。vivian4123 (2013-10-06 15:43:47)
相关疾病：
口蹄疫
你可以多换几种载体试一下啊，我觉得蛋白表达是一个很奇怪的东西，要多是几个载体，说不定哪一个就可以了，你可以试一下有trx或gst等促进蛋白可溶性表达的载体，pet32a既有trx，又有his-tag，方便纯化，可以试一下。还有已从方法就是先查一下自己的蛋白的性质，及等电点，然后根据这个调pbs的PH,有时候也能把蛋白调到上清里面。但这种方法不能保证蛋白的结构是否还有，因为我表达的是抗原，所以有线性表位就可以了，没有关注它是否有二三级的结构
（我看你是做口蹄疫的，如果做得是诊断的话，这种方法是可行的，直接拿调过pH值得pbs来融沉淀，蛋白纯度还是会挺高的，有时候可以不用再纯化的）。
wzqzy (2013-10-06 15:44:11)
QUOTE:
因为我表达的蛋白需要测酶活性，所以必须在上清里，没有试过哺乳动物细胞，老板说我的蛋白很好表达，原核表达就行了，我也很无语啊
remenb (2013-10-06 15:44:51)
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首先应该弄明白这个载体上的片断有没有优化，验证过，是不是真的可以做到可溶表达。很多实验室的质粒传来传去最后发生了突变也不知道，这种事情不少。
我经常纯化重组表达的HRV3C蛋白酶，确实很容易，低温诱导+注意防止氧化就行了。你是怎么操作的？
最笨的一个办法就是37度摇菌过夜，第二天加1mM IPTG，16-18度摇4小时后收菌，多少都应该有一点可溶表达的。
damingxia0904 (2013-10-06 15:45:17)
FMDV的3C蛋白酶表达，文献上已经有人做过了。 cuturl('http://scripts.iucr.org/cgi-bin/paper?en5101')
cuturl('http://jvi.asm.org/content/81/1/115.full?view=long&pmid=17065215')
cuturl('http://upetd.up.ac.za/thesis/available/etd-08042008-104418/')
增强蛋白质可溶性表达的策略：
Improving Protein Solubility
In many cases the expressed protein is insoluble and accumulates in so-called inclusion bodies. This is especially true under conditions of high level expression. Several strategies are available to improve the solubility of the expressed protein.
Reducing the rate of protein synthesis.
This can be done by:
lowering the growth temperature. This decreases the rate of protein synthesis and usualy more soluble protein is obtained.
using a weaker promoter (e.g. trc instead of T7).
using a lower copy number plasmid.
lowering the inducer concentration.
Changing the growth medium:
addition of prostethic groups or co-factors which are essential for proper folding or for protein stability.
addition of buffer to control pH fluctuation in the medium during growth.
addition of 1% glucose to repress induction of the lac promoter by lactose, which is present in most rich media (such as LB, 2xYT).
addition of polyols (e.g. sorbitol) and sucrose. The increase in osmotic pressure caused by these additions leads to the accumulation of osmoprotectants in the cell, which stabilize the native protein structure.
addition of ethanol, low molecular weight thiols and disulfides, and NaCl.
(Georgiou, G. & Valax, P. (1996) Current Opinion Biotechnol. 7, 190-197)
Co-expression of chaperones and/or foldases.
Two classes of proteins play an important role in in vivo protein folding.
Molecular chaperones promote the proper isomerization and cellular targeting by transiently interacting with folding intermediates. The best characterized E. coli systems are:
GroES-GroEL
DnaK-DnaJ-GrpE
ClpB
Foldases accelerate rate-limiting steps along the folding pathway. Three types of foldases play an important role:
peptidyl prolyl cis/trans isomerases (PPI's)
disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC)
protein disulfide isomerase (PDI) - an eukaryotic protein that catalyzes both protein cysteine oxidation and disulfide bond isomerization. It also exhibits chaperone activity.
Co-expression of one or more of these proteins with the target protein could lead to higher levels of soluble protein. The levels of co-expression of the different chaperones/foldases have to be optimized for each individual case. DsbA and DsbC have also shown possitive effects on expression levels when used as a fusion partner.
Periplasmic expression:
Secretion of the target protein to the periplasm has a number of distinct advantages:
the oxidizing environment of the periplasm allows for the formation of disulfide bonds, which does not occur in the reducing environment of the cytoplasm.
the periplasm contains two foldases, disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC), that catalyze the formation and isomerization of disulfide bonds.
reduced proteolysis (since less proteins are present).
allows for the accumulation of proteins that are toxic in the cytoplasm.
engineering of an authentic N-terminus.
damingxia0904 (2013-10-06 15:45:36)
Using specific host strains:
The solubility of disulfide bond containing protein can be increased by using a host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available (Novagen):
AD494, which has a mutation in thioredoxin reductase (trxB).
Origami, a double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor).
Addition of a fusion partner:
Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
Expression of a fragment of the protein:
E. coli does not express well very large proteins (> 70 kDa). Chosing a smaller fragment of the target protein can improve expression levels and solubility.
The solubility of a poorly soluble (or insoluble) protein can also be improved by selecting only a soluble domain for expression.
In vitro denaturation and refolding of the protein:
When despite all efforts the target protein still is expressed in inclusion bodies, then the last resort is to denature and refold the protein in vitro. This procedure is carried out in three phases:
isolation of the inclusion bodies.
solubilization and denaturation of the target protein. This is done by the addition of a denaturing agent (usually guanidine or urea) under reducing conditions (e.g. 20 mM DTT).
refolding of the protein by removing the denaturating agent using dialysis, dilution or chromatography. For proteins containing disulfide bonds this has to be carried out in the presence of a redox shuttling system e.g. reduced and oxidized glutathione.
cuturl('http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/improving_protein_solubility/')
tuomu45 (2013-10-06 15:46:14)
（我看你是做口蹄疫的，如果做得是诊断的话，这种方法是可行的，直接拿调过pH值得pbs来融沉淀，蛋白纯度还是会挺高的，有时候可以不用再纯化的）。
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我表达的蛋白是用来做药物筛选的，所以需要上清表达，并且要高浓度。这就增加得蛋白表达得难度啊，、、
我们实验室就那几种载体，多数是PET系列的，32a貌似也有可以用来试一下，，
我想请教一下，知道蛋白质的等电点后，要将pbs调到什么程度容易上清表达呢
tuomu45 (2013-10-06 16:00:22)
我经常纯化重组表达的HRV3C蛋白酶，确实很容易，低温诱导+注意防止氧化就行了。你是怎么操作的？
最笨的一个办法就是37度摇菌过夜，第二天加1mM IPTG，16-18度摇4小时后收菌，多少都应该有一点可溶表达的。
===========================================================================================================
我是先37度摇菌复苏8个小时，在从复苏菌中取出定量菌活化两个小时，待OD值达到0.6左右加IPTG诱导，18度诱导是十几个小时、、、，你说的防止氧化要怎么做呢？？还有如果摇菌过夜后第二天加IPTG，OD值会不会太高呢，期待回复，谢谢啦
tuomu45 (2013-10-06 16:01:19)
cuturl('http://jvi.asm.org/content/81/1/115.full?view=long&pmid=17065215')
cuturl('http://upetd.up.ac.za/thesis/available/etd-08042008-104418/')
增强蛋白质可溶性表达的策略：
Improving Protein Solubility
In many cases the expressed protein is insoluble and accumulates in so-called inclusion bodies. This is especially true under conditions of high level expression. Several strategies are available to improve the solubility of the expressed protein.
Reducing the rate of protein synthesis.
This can be done by:
lowering the growth temperature. This decreases the rate of protein synthesis and usualy more soluble protein is obtained.
using a weaker promoter (e.g. trc instead of T7).
using a lower copy number plasmid.
lowering the inducer concentration.
Changing the growth medium:
addition of prostethic groups or co-factors which are essential for proper folding or for protein stability.
addition of buffer to control pH fluctuation in the medium during growth.
addition of 1% glucose to repress induction of the lac promoter by lactose, which is present in most rich media (such as LB, 2xYT).
addition of polyols (e.g. sorbitol) and sucrose. The increase in osmotic pressure caused by these additions leads to the accumulation of osmoprotectants in the cell, which stabilize the native protein structure.
addition of ethanol, low molecular weight thiols and disulfides, and NaCl.
(Georgiou, G. & Valax, P. (1996) Current Opinion Biotechnol. 7, 190-197)
Co-expression of chaperones and/or foldases.
Two classes of proteins play an important role in in vivo protein folding.
Molecular chaperones promote the proper isomerization and cellular targeting by transiently interacting with folding intermediates. The best characterized E. coli systems are:
GroES-GroEL
DnaK-DnaJ-GrpE
ClpB
Foldases accelerate rate-limiting steps along the folding pathway. Three types of foldases play an important role:
peptidyl prolyl cis/trans isomerases (PPI's)
disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC)
protein disulfide isomerase (PDI) - an eukaryotic protein that catalyzes both protein cysteine oxidation and disulfide bond isomerization. It also exhibits chaperone activity.
Co-expression of one or more of these proteins with the target protein could lead to higher levels of soluble protein. The levels of co-expression of the different chaperones/foldases have to be optimized for each individual case. DsbA and DsbC have also shown possitive effects on expression levels when used as a fusion partner.
Periplasmic expression:
Secretion of the target protein to the periplasm has a number of distinct advantages:
the oxidizing environment of the periplasm allows for the formation of disulfide bonds, which does not occur in the reducing environment of the cytoplasm.
the periplasm contains two foldases, disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC), that catalyze the formation and isomerization of disulfide bonds.
reduced proteolysis (since less proteins are present).
allows for the accumulation of proteins that are toxic in the cytoplasm.
engineering of an authentic N-terminus.
Secretion is achieved by the addition of a leader sequence (signal peptide) to the N-terminus of the target protein. Most used leader sequences are pelB and ompT. Unfortunately, expression yield are usually much lower and not all expressed protein is secreted into the periplasm but is also found in the medium, the cytoplasm and the cytoplasmic membrane.
Using specific host strains:
The solubility of disulfide bond containing protein can be increased by using a host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available (Novagen):
AD494, which has a mutation in thioredoxin reductase (trxB).
Origami, a double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor).
Addition of a fusion partner:
Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
Expression of a fragment of the protein:
E. coli does not express well very large proteins (> 70 kDa). Chosing a smaller fragment of the target protein can improve expression levels and solubility.
The solubility of a poorly soluble (or insoluble) protein can also be improved by selecting only a soluble domain for expression.
In vitro denaturation and refolding of the protein:
When despite all efforts the target protein still is expressed in inclusion bodies, then the last resort is to denature and refold the protein in vitro. This procedure is carried out in three phases:
isolation of the inclusion bodies.
solubilization and denaturation of the target protein. This is done by the addition of a denaturing agent (usually guanidine or urea) under reducing conditions (e.g. 20 mM DTT).
refolding of the protein by removing the denaturating agent using dialysis, dilution or chromatography. For proteins containing disulfide bonds this has to be carried out in the presence of a redox shuttling system e.g. reduced and oxidized glutathione.
cuturl('http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/improving_protein_solubility/')
===============================================================
非常感谢管理员提供的信息，很有用。
就你提到的那几篇文献我也有看过，但用相同得条件我的就是不表达，很奇怪啊
ending (2013-10-06 16:03:22)
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确保你的亚克隆读码框正确。
另外，建议你写信向人间直接索取构建好的质粒，不需要浪费这样不必要的时间了。
831226 (2013-10-06 16:03:53)
我们实验室就那几种载体，多数是PET系列的，32a貌似也有可以用来试一下，，
我想请教一下，知道蛋白质的等电点后，要将pbs调到什么程度容易上清表达呢，
===========================================================================================================
溶液的pH 低于或高于蛋白质的pI 都有利于蛋白质水溶性的增加，不想预测pI的话，你也可是试着往两边都加一下试试，慢慢的加，看加到什么程度能融，具体要偏酸还是偏碱最好根据你将来纯化时的溶液pH来定，比如说你要用镍柱纯化，咪唑的pH可能要求8.0。蛋白浓度若是纯化下来不高的话也可以浓缩啊，我觉得用冻干机浓缩还不错啊。
wzqzy (2013-10-06 16:04:30)
另外，建议你写信向人间直接索取构建好的质粒，不需要浪费这样不必要的时间了。
=============================================================================
额，这个有点困难吧，别人怎么会把构建好的质粒给我呢？？呵呵我再想想办法吧，谢谢你的宝贵意见
kswl870 (2013-10-06 16:05:05)
不要怕OD值高，试一试吧
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提纯的时候加10mM巯基乙醇就可以防止氧化了，前提是你的蛋白本身结构里不存在二硫键。如果有的话可以用胱氨酸或者谷胱甘肽。巯基乙醇容易挥发所以久存后要适当添加。过镍柱前必须稀释，防止还原柱上的金属离子。
【讨论帖】蛋白要怎么优化条件才能上清表达？