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Toxic Protein
 

Toxic protein cloning and expression

Contents of toxic protein cloning and expression

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Introduction of protein toxicity

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Why a protein is toxic to a host cell?

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What percentage of protein is toxic?

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Protein toxicity effects

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Are recombinant DNA and RNA toxic to the host cells?

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The phenotypes of protein toxicity in molecular biology studies

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Current technologies of toxic protein cloning and expression

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New technologies of toxic protein cloning and expression

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The growth media is critical for toxic protein cloning and expression

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Transcription repressors expressed from expression vector and host cell for toxic protein cloning and expression

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Transcription terminator and multiple operators on expression vector for toxic protein cloning and expression

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Expression vectors with multiple operators for toxic protein cloning and expression

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Development of the technologies of toxic protein cloning and expression

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Re-invented IPTG-inducible system for toxic protein cloning and expression

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Classification of protein toxicity by DNA cloning and protein expression

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Toxic protein cloning and expression strategies

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Other techniques may be easily optimized for toxic protein cloning and expression

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Other molecular biology technologies for toxic protein cloning and expression

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Advantage of protein toxicity

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Summary of technologies for toxic protein cloning and expression

Introduction of protein toxicity

Since the beginning of recombinant DNA technology in early 1970s, scientists have found many structural genes or protein-coding cDNAs cannot be readily cloned into a plasmid with a consecutively expressing promoter. With introduction of some regulatory DNA sequences to shut down the consecutive expression of the proteins, most structural genes can be cloned and some of them can be expressed. This leads to the development of IPTG induction system. The lac operon is one of the most studied regulatory genes. The lacI repressor binding site is termed lac operator. With introduction of a lac operator after a promoter, lacI repressor will bind on the operator and reduce the non-induced transcription over 90%. This technology leads to the cloning and expression of many genes and proteins. It is the most commonly used system in molecular biology studies.

Why a protein is toxic to a host cell?

In a biological system, a particular protein is expressed only in a specific subcellular location, tissue or cell type, during a defined time period, and at a particular quantity level. This is the so called spatial, temporal, and quantitative expression. Recombinant protein expression often introduces a foreign protein in a host cell and expresses the protein at levels significantly higher than the physiological level of the protein in its native host and at the time the protein is not needed. The over-expressed recombinant protein will perform certain function in the host cell if the protein is expressed soluble and functional. The function of the expressed recombinant protein is often not needed by the host cell. In fact the function of the protein may be detrimental to the proliferation and differentiation of the host cell. The observed phenotypes of the host cells are slow growth rate and low cell density. In some cases, the recombinant protein causes death of the host cell. These phenomena are described as protein toxicity. These recombinant proteins are called toxic proteins.

What percentage of protein is toxic?

Protein toxicity is a commonly observed phenomenon. All active proteins will perform certain functions. All these functions with few exceptions are needed by the host cells and therefore they interfere with cellular proliferation and differentiation. The appeared phenotype of the effects of these proteins to the host cells is their "toxicity". The phenotypes of the protein toxicity are listed bellow. We estimated that about 80% of all soluble proteins have certain degree of toxicity to their hosts. About 10% of all proteins are highly toxic to host cells. The completely insoluble or dysfunctional proteins will not be toxic to the host cell, though they may drain the cellular energy to produce them when over-expressed. Protein over-expression creates metabolic burden for the host cell, but this burden is not toxicity to the cell. Some low solubility or partially functional proteins may still be toxic to the host.

Protein toxicity effects

Protein toxicity adversely affects the cloning and expression. For example, the genes or cDNAs of the most toxic proteins are difficult to be cloned. Protein toxicity is the most important reason for DNA cloning or subcloning problems. Most expression problems are also the result of protein toxicity. With optimization of expression vectors and host cells, we estimate that 80% protein yield problems are the caused by protein toxicity. Only 20% yield problems are the results of other reasons such as codon usage. This is why many codon optimized genes still have expression problems.

Are recombinant DNA and RNA toxic to the host cells?

A recombinant DNA fragment is normally not toxic to the host cell unless it contains repetitive sequences which have high affinity to some transcription factors. A functional recombinant RNA can be toxic to the host cells. We estimated that over 80% host toxicity are caused by recombinant proteins, about 15% are caused by recombinant RNA, and less than 1% are caused by recombinant DNA. To reduce or eliminate the toxicity by recombinant RNA, the recombinant RNA transcription should repressed until it is needed. To prevent the toxicity by recombinant DNA, the recombinant DNA fragment should be cloned in the site flanked by some repressors such as the vectors with multiple operators listed bellow. These vectors will bind to repressors to make the DNA fragment inaccessible to the cellular factors and therefore eliminate the toxicity.

The phenotypes of protein toxicity in molecular biology studies

bulletToxic protein cloning problems
bulletNo colony in cloning
bulletFewer colonies than those in regular cloning experiments
bulletSmall percentage of positive clones
bulletWrong orientation
bulletMutations leading to defective products or no expression
bulletToxic protein expression problems
bulletNo expression
bulletLow yield
bulletDefective proteins
bulletInconsistent expression
There is expression sometimes while other times there is no expression. The expression seems inconsistent or unpredictable.
bulletToxic protein host cell growth rate and cell density problems
bulletCell grows significantly slower and cannot reach its normal density in a given medium before induction.
bulletCell grows significantly slower and cannot reach its normal density after induction.
bulletGrowth rate and cell density are low in normal strain. In contrast, the growth rate and cell density are close to normal in a detoxification cell strain. This also indicates the detoxification cell strain will likely to clone and express the target protein.
bulletTransformation efficiency problems
bulletThe transformation efficiency of the vector containing a toxic protein is lower or significantly lower than the control vectors or the vectors containing non-toxic proteins.
bulletTransformation efficiency in a normal cell strain is significantly lower than that in a detoxification strain. This observation also indicates that the detoxification strain will likely to clone and express the target protein.

Current technologies of toxic protein cloning and expression

After the development of IPTG-inducible system, many proteins are still difficult to be cloned and expressed. Arabinose and rhamnose inducible systems appear to be tightly regulated expression systems. The protein yield in arabinose system is low. Rhamnose is uncommon sugar. Both systems are not as widely used as the IPTG-inducible system. T7 lysozyme is used to repress T7 RNA polymerase in pLysS and pLysE strains. Since lysozyme itself is toxic to the cells at high concentration, ideal repression is not achieved with these strains. In addition, T7 lysozyme will not inhibit E.coli RNA polymerase, and it is not useful for the promoters other than T7. Another idea is to screen the cell strains that will clone and express a particular toxic protein. Cell strains such as C41 and C43 are generated by this strategy. Clearly these cell strains are suited only for some proteins or group of proteins that have been screened for. A more successful strategy is to introduce exogenous lacI repressor to the host cells by inserting lacIq gene on the expression vector or on a compatible plasmid in a host cell. This strategy proves to be good in cloning and expressing many toxic proteins, but is still insufficient for the highly toxic proteins. The lacI level produced by the compatible plasmid, usually based on pACYC vectors, is still insufficient to repress the promoter on a higher copy number plasmid based pBR322 or pUC. Therefore some extremely toxic proteins still cannot be cloned or expressed in this system. After these toxic proteins are cloned, many of them are difficult to express at sufficient level for biochemical studies.

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New technologies of toxic protein cloning and expression

The growth media is critical for toxic protein cloning and expression

The following is a simplified binding equilibrium between lacI repressor (r stands for the repressor binding with inducer which cannot bind to the operator and R stands for the repressor without inducer which has high affinity to the operator to repress the transcription) and its binding site (operator O).

If the cDNA is often cloned in a wrong orientation, there are many mutations in the cloned constructs, or the cDNA cannot be cloned at all, the protein encoded by the cDNA is toxic to the host cell. Three strategies may be used to reduce protein toxicity. The first and easiest way is to use a culture medium that can reduce leaky expression. Our DetoXTM medium contains high concentration of transcription inhibitors. Regular media such as LB or TB contain no inhibitors for transcription. As a matter of fact, most commonly used rich media are made from enzymatic digestion of cow milk protein casein (tryptone or peptone). Casein inevitably contains lactose. Experiments indicate that even trace amount of lactose in casein peptone can induce protein expression. Lactose presented in growth media is the number one culprit for protein toxicity. There are two ways to solve the problem. One is to prepare the medium with no lactose at all. The other is to add transcription inhibitors in the medium. E.coli cells prefer glucose as a carbon or energy source. In the presence of glucose, E.coli cells will not use lactose. Therefore glucose serves as an inhibitor for IPTG-inducible promoters (it also inhibits arabinose promoter) and lac operon will not be induced. Our DetoXTM medium contains no lactose at all.  Instead it contains high concentration of glucose which will support high density E.coli growth (OD600 > 30) in a shake flask container. The high cell density is important to increase the yield of the toxic protein.

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Transcription repressors expressed from expression vector and host cell for toxic protein cloning and expression

A second way to help with expression is to use cell strains containing sufficient amount of transcription repressors to repress the inducible promoter. The endogenous lacI expression in E.coli cells is not sufficient to repress the leaky expression of currently used expression vectors. Introducing exogenous lacI repressor to the host cells by inserting lacIq gene on the expression vector or on a compatible plasmid in a host cell is successful. This strategy proves to be good in cloning and expressing many toxic proteins, but is still insufficient for the highly toxic proteins. The lacI level produced by the compatible plasmid, usually based on pACYC vectors, is still insufficient to repress the promoter on a higher copy number plasmid based pBR322 or pUC. Therefore some extremely toxic proteins still cannot be cloned or expressed in this system. We re-engineer the compatible plasmids that have significant higher copy number than pBR322 to solve this problem. Therefore our detoxification strains express three to five times higher amount of lacI than regular lacI strains.

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Transcription terminator and multiple operators on expression vector for toxic protein cloning and expression

Most commercially available expression vectors contain only one lacI repressor binding site (operator O2). All these vectors have about 5% leaky expression. Many of these vectors do not contain transcription terminators upstream of the inducible promoter.

We introduced a strong transcription terminator up stream of the inducible promoter and two to three operators or repressor binding sites on our expression vectors by mimicking lac operon. No more leaky expression can be detectable by western analysis when our detoxification media and cell strains are used in combination. All toxic proteins tested can be successfully cloned and expressed using our detoxification media, cell strains, vectors or combination of them.

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Expression vectors with multiple operators for toxic protein cloning and expression

Development of the technologies of toxic protein cloning and expression

In the process of cloning and expressing nuclear hormone receptors, we found some of the receptors are toxic to the E.coli hosts. Some of the full-length nuclear hormone receptor pairs such as human RXRα and RARβ are so toxic to the host that we could not clone them in any of the tested vectors or cell strains, including pGEX, pET, and pBAD vectors or plysS, placI, and C41 cell strains.

After careful examination of the natural lac operon, we found reports of three lac operators (repressor binding sites) flanking the native inducible promoter. Most existing IPTG-inducible vectors contain only one operator. These vectors have about 5% leaky expression. It is the leaky expression that prevents highly toxic proteins to be cloned and expressed in these vectors and cell strains.

After engineering our vectors with multiple operators and a strong terminator upstream of the inducible promoter, we successfully cloned the full-length receptor pairs in our expression vectors. However cell strains with these cloned vectors could not reach inducible cell density (OD600). Next we cloned lacIq gene in a pACYC-based vector containing a different selection marker. Co-expression of lacIq gene with the receptors pairs increased the cell density. The cell density was still low, leaky expression was detected by Western analysis, and no significant amount of protein could be purified. Commonly used pACYC vectors are low copy number plasmids (about 10 copies per cell), lacI repressors produced by these vectors may not be sufficient to bind operators on high copy number expression plasmids based pUC plasmids (more than 200 copies per cell). We re-engineered lacI expression vectors based on pACYC into medium to high copy number plasmids (about 50 to 100 copies per cell). We also re-engineered compatible cell strains expressing different levels of lacI repressor. When a low copy number plasmid based on pBR322 containing the receptor pairs transformed in a high level lacI strain, E.coli cells can reach normal cell density and no more leaky expression can be detected with Western analysis. However, little protein can be purified since cell density decreases rapidly after induction when we used common media such as LB or TB.

Next we formulated high density growth media which contain transcription inhibitors and can support high density cell growth. E.coli cells can reach a density of OD600 > 30 in these special media in shake flask containers. Combining low copy number expression vector with multiple operators and high level lacI strain with a high density growth medium, we successfully cloned, expressed and purified the most toxic receptor pairs with reasonable yield. Many other toxic proteins can be cloned and expressed with these vectors, cell strains and media. Since most proteins are not as toxic as the tested receptor pair, they only need vectors, cell strains, media or combination of two of them to achieve successful cloning and expression. Over two hundred receptor,  chaperone and enzyme constructs have been tested so far. Eighty percent of these proteins present certain degree of toxicity to their host. About 10% of these proteins are highly toxic and have cloning, expression, or cell growth problems. To our delight, all of these toxic proteins can be cloned and expressed in our re-invented IPTG inducible system.

Re-invented IPTG-inducible system for toxic protein cloning and expression

  1. A strong transcription terminator is placed immediate upstream of the inducible promoter to prevent transcription "read-through" from upstream real or cryptic promoters.

  2. Multiple repressor binding sites or operators are engineered flanking the inducible promoter resembling those on native lac operon. This increase the transcription repression from about 95% to over 99.9%.

  3. lacI transcription repressor is over expressed both on the expression vectors and in the host cell strains to increase the available repressor concentration.

  4. Low copy number expression vector and high copy number lacI plasmid in the host cell are used for most toxic proteins.

  5. Transcription inhibitors are included in the growth media to reduce or eliminate un-induced or leaky expression.

  6. High density growth media are formulated to support E.coli growth to OD600 = 30 - 50 to increase protein yield.

Classification of protein toxicity by DNA cloning and protein expression

We classify the protein toxicity into different degrees according their clonability and expression levels.

  1. The gene or cDNA encode the protein cannot be cloned

  2. No detectable expression

  3. Expression level is very low (in micrograms per liter culture)

  4. Expression level is low (in hundreds of micrograms per liter culture)

  5. Expression level is reasonable but can be improved (from hundreds of micrograms to milligrams per liter)

  6. No toxicity (tens to hundreds of milligrams per liter culture)

The first degree is the most toxic, while the sixth degree is not toxic.

Toxic protein cloning and expression strategies

Once the toxicity of a protein is determined, different strategies should be used to express proteins with different degree of toxicity. The most toxic proteins will need combination of vectors, cell strains and media to achieve successful cloning and expression. The least toxic protein may only require any one of these products.

  1. The gene or cDNA encode the protein cannot be cloned
    These are the most toxic proteins. They may need combination of new detoxification vectors, cell strains and high density growth media to achieve successful cloning and expression.

  2. No detectable expression
    These are highly toxic proteins. They will need both our detoxification cell strains and high density growth media for cloning and expression. Some of them may also need the tightly regulated vectors with multiple repressor binding sites.

  3. Very low yield (in micrograms per liter)
    These proteins will need new cell strains and high density growth media to achieve successful expression.

  4. Low yield (in hundreds of micrograms per liter)
    These proteins are moderately toxic. They may need either detoxification cell strains or high density growth media to achieve successful expression. Combination of our cell strains and media can increase yield significantly.

  5. Yield OK but can be improved (from hundreds of micrograms to milligrams per liter)
    Using our media alone can increase protein yield significantly. Using ETI's cell strains alone can also increase these protein yields.

  6. No toxicity
    Since high density growth media can support cell density to OD600 = 30, they can increase non-toxic protein yield five to twenty times over common media such as LB or TB.

Hundreds of toxic proteins were cloned and expressed with combination of above technologies. There have been no exceptions including the most toxic protein. These technologies can be easily used in most labs except the expression vectors which need subcloning. Expression vectors involving subcloning are needed only for the most toxic proteins. Low and moderately toxic proteins can be cloned and expressed with the high density growth media and cell strains.

Other techniques may be easily optimized for toxic protein cloning and expression

These technologies may be used in most molecular biology labs.

bulletInduction conditions
bulletInduction time
Induction time should be closely monitored to produce reasonable amount of protein while the host cells are not devastated (protein degradation or cell death). Monitoring induction time is also important for some unstable proteins.
bulletInducer concentration
Inducer concentration is directly related to protein yield and therefore related to protein toxicity. In many cases lower inducer concentration not only results in higher protein yield but also lower protein toxicity.
bulletInduction temperature
Many proteins are active at 37 0C. At lower temperature, these proteins may be less active or lose activity. Therefore their toxicity may be reduced or eliminated at lower temperature. The activities of some proteins decrease with lowering the temperature. Other proteins may lose their activities at a temperature point. The minimum temperature E.coli can proliferate and differentiate (slowly) appears to be at about 10 0C. Lower temperature also results in increased protein solubility. Longer induction time should always be associated with lower temperature because of lower protein synthesis rate. Temperatures of 25 and 15 degree Celsius are commonly used for induction of toxic proteins.
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Inhibitor in medium
It has been well established that E.coli cells prefer glucose as carbon or energy source. In the presence of glucose, E.coli will not use lactose or arabinose and these inducible promoters will not be induced. About 0.5% glucose may be added to a common medium to inhibit related leaky expression. With inhibitors in media, some low toxic proteins can be cloned. However inhibitors in medium alone are not sufficient for most toxic protein production. The yields of most toxic proteins are not sufficient for biochemical studies. High density growth media or fermentation will be needed for reasonable yield.

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High concentration of antibiotics
We do not find this strategy effective in toxic protein expression in our hands, although it was reported working for some proteins.

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Plating inoculation
This strategy has been reported to be successful for some proteins. But it will not work for the proteins that cannot be cloned or grow into screenable colonies.

bulletOther detoxification strains
bulletC41 and C43 strains appear to be useful in some toxic proteins especially some membrane proteins. Cell strains such plysS and plysE are not as good as placI strain. They may be used when no other detoxification strains are available. Commonly used placI strain express lacI repressor on a low copy number plasmid. Its lacI level may not be sufficient for many toxic protein expressions. Our lacI strains express different levels of lacI and can meet different toxic protein expression needs.
bulletT7 lysozyme is used to repress T7 RNA polymerase in pLysS and pLysE strains. Since lysozyme itself is toxic to the cells at high concentration, ideal repression is not achieved with these strains. In addition, T7 lysozyme will not inhibit E.coli RNA polymerase, and it is not useful for the promoters other than T7.
bulletOther commercial available lacI strains. These lacI strains often give better results than those may be obtained from pLysS and pLysE strains since lacI repressor has no apparent effect on host cells. However these lacI repressors are all expressed on low copy number pACYC based plasmids. The lacI repressors expressed may not be sufficient to repress the most toxic proteins.

These technologies are generally good for proteins of low toxicity and for some moderately toxic proteins. Highly toxic proteins cannot be cloned or expressed with these technologies. In addition, the expression levels or yields of the toxic proteins are often low and may not be sufficient for some biochemical studies.

Other molecular biology technologies for toxic protein cloning and expression

Other technologies may also be used in addition to or in combination of above technologies to improve cloning efficiency and protein yield.

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Low copy number plasmid
Lower copy number plasmids have lower protein yield and are better regulated. Therefore they are better in cloning and expressing toxic proteins than high copy number plasmids. In this case less is more. This is also true for weak promoters.

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Tightly regulated vector
Commonly used plasmid vectors such as pGEX and pET contain only one lac operator downstream of their promoters and have about 5% leaky expression. Our vectors mimics native lac operon and contain two to three lac operators flanking their inducible promoters. Combining with our lacI cell strains and special media, leaky expression is not detectable by Western analysis.

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Weak promoter
Strong promoters have high protein yield and high leaky expression before induction. Therefore toxic proteins appear more toxic under these promoters than the same proteins under weak promoters.

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Strong terminator upstream of inducible promoter
A strong transcription terminator upstream of inducible promoter will prevent transcription read-through from upstream real and cryptic promoters. All of our expression vectors contain strong terminators upstream of inducible promoter. It is useful to keep in mind when constructing an expression vector.

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Fusion expression
Large fusion partners such as GST or Trx will reduce protein toxicity in some cases. Small tags such as his-tag will not alter toxicity property significantly. In rare cases fusion partner can increase protein toxicity.

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Individual domains
Individual domains are almost always less toxic than the full-length proteins. Sometimes individual domains may lose toxicity completely and therefore are not functional. Other strategies have to be used if individual domains are highly toxic.

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Different cellular compartment
If cytoplasmic expression is toxic, secretory expression may reduce or eliminate toxicity.

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Insoluble protein
Insoluble proteins are not functional, therefore they are not toxic. Fusion with some proteins to produce insoluble protein can be a good strategy if refolding can be easily achieved.

These Technologies require some molecular biology experiments. In some cases, the experiments can be extensive. In addition, the results are often hit or miss. The outcomes cannot be predicted. Many of above technologies when used individually do not work for the most toxic proteins in cloning or expression.

Advantage of protein toxicity

Almost everything has two sides. Toxic proteins are no exception. The reason that these proteins are toxic is that they are soluble and functional. All highly toxic proteins we tested, when successfully expressed, are soluble and functional.

Summary of technologies for toxic protein cloning and expression

A biological system as simple as E.coli is an integral system. Protein expression can be regulated at different levels. The most import controls of many proteins appear to be at transcription level. The nature of the expression vectors, the cell strains, and the growth media are all involved in the transcription regulation. Even when a tightly regulated vector and a cell strain with high level of repressor are used, it is still important to make sure that there is no inducer in the culturing medium. Trace amount of inducer such as lactose presented in tryptone or peptone can induce expression in some commonly used vectors and cell strains. Medium is one of the most important factors in toxicity concerns. Medium is necessary but not sufficient for highly toxic protein expression. Tightly regulated vectors and cell strains with reasonable amount of repressors will be needed for highly toxic protein expression. The technologies presented here represent the most advanced toxic protein cloning and expression system up to date. With combination of above technologies, all toxic proteins tested can be successfully cloned and expressed at reasonable levels. The expression vector, cell strain, and medium combination may present a general strategy in toxic protein expression in higher organisms such yeast, insect, and mammal.

Related literatures of toxic protein cloning and expression

Bacterial growth media
Plasmid DNA yield
Protein yield
Protein solubility

Related products of toxic protein cloning and expression

Bacterial E.coli growth media
DNA ladders or DNA markers
Expression vectors
Competent cells for cloning and expression
E.coli cell strains for protein expression

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