His tag protein purification | Application of his tag purification | Affinity chromatography

Summary

This video script delves into His-tag protein purification, a method utilizing single-step affinity chromatography with a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The technique leverages the specific interaction between the matrix and a hexahistidine tag attached to the protein of interest, enabling efficient separation from a protein mixture. The process involves equilibration, binding, washing, and elution steps, ensuring the purification of the native protein. Advantages include compatibility with cell culture media, ease of use, sensitivity, and cost-effectiveness, though some optimization may be necessary to minimize non-specific binding.

Takeaways

• 🧪 His-tag protein purification utilizes a single-step affinity chromatography method with immobilized metal ion chromatography (IMAC).
• 🔗 The purification relies on the specific interaction between a nickel-nitrilotriacetic acid (Ni-NTA) matrix and a hexahistidine tag attached to the protein of interest.
• 🔬 The hexahistidine tag is incorporated at the DNA level, either at the N-terminal or C-terminal of the open reading frame, and then expressed in bacterial or mammalian cells.
• 📦 The workflow of His-tag purification includes equilibration, binding, washing, and elution steps to separate the tagged protein from a mixture.
• 🌡️ Equilibration buffer must be compatible with the protein of interest, considering factors such as ionic strength and pH to maintain protein stability.
• 🔒 During the binding step, the tagged protein binds to the Ni-NTA matrix through non-covalent interactions like coordinate bonds, while other proteins in the lysate do not bind.
• 🚿 The washing step removes non-specifically bound proteins, ensuring that only the specifically bound tagged protein remains on the column.
• 🌟 The elution step involves using a buffer with altered pH and ionic strength to release the bound protein, which can then be collected.
• 🛑 Post-purification, techniques like SDS-PAGE and Western blot can be used to verify the success of the purification process.
• 💰 His-tag purification is cost-effective compared to other chromatographic techniques such as gel filtration or HPLC.
• ⚙️ Optimization may be required to minimize non-specific binding, which involves adjusting the ionic strength of the buffers used in the process.
• 📚 Additional resources and flashcards on this topic can be found on the instructor's social media platforms, with links provided in the video description.

Q & A

What is His-tag protein purification?

His-tag protein purification is a method that allows for the single-step purification of proteins using immobilized metal ion affinity chromatography with a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The protein is tagged with hexahistidine, which interacts specifically with the matrix.

How is the His-tag attached to the protein?

The His-tag is attached to the protein by cloning the open reading frame with a hexa-histidine tag at the N-terminal or C-terminal into an expression vector, which is then introduced into bacterial or mammalian cells to produce the tagged protein.

What is the principle behind the separation in His-tag protein purification?

The principle behind the separation is the specific interaction between the hexahistidine tag on the protein and the nickel ions on the Ni-NTA matrix, which involves non-covalent interactions like coordinate bonds.

What are the steps involved in the His-tag protein purification process?

The steps are equilibration, binding, washing, and elution. Equilibration involves running a buffer through the column, binding is where the tagged protein attaches to the matrix, washing removes non-specific interactions, and elution collects the purified protein.

Why is the equilibration buffer important in the purification process?

The equilibration buffer is important because it ensures that the column material is soaked and equilibrated at a specific pH that is compatible with the protein of interest, preventing denaturation.

What factors should be considered when choosing the equilibration buffer?

Factors to consider include ionic strength and pH, as these can affect protein stability and prevent denaturation during the purification process.

How does the binding step in His-tag purification work?

In the binding step, the protein sample, usually a cell lysate, is loaded onto the column. The tagged protein binds to the Ni-NTA matrix through non-covalent interactions, specifically coordinate bonds.

What is the purpose of the washing step in the purification process?

The washing step ensures that any non-specific, untagged proteins are removed from the column, breaking down weak bonds and retaining only the specific interactions between the His-tag and the matrix.

How is the elution step different from the other steps in His-tag protein purification?

The elution step involves using a buffer with altered pH and ionic strength to loosen the binding between the matrix and the protein, allowing the purified protein to be collected.

What methods can be used to verify the success of His-tag protein purification?

SDS-PAGE and Western blot can be used to verify the purification by checking the presence and purity of the protein of interest.

What are some benefits of using His-tag protein purification compared to other techniques?

Benefits include compatibility with cell culture media and lysates, ease of use, sensitivity, specificity, and being more cost-effective compared to techniques like gel filtration chromatography or HPLC.

What are some potential disadvantages of His-tag protein purification?

Disadvantages include the need for optimization to minimize non-specific binding, which may require calibration of the ionic strength of the equilibration or binding buffer.

Outlines

00:00

🔬 His Tag Protein Purification Method

This paragraph introduces His tag protein purification, a single-step affinity chromatography technique for protein separation using a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The process hinges on the specific interaction between the matrix and a hexahistidine tag attached to the protein of interest, facilitated by coordinate bonds. The method begins with cloning a hexa-histidine tag into an expression vector at the protein's N- or C-terminus. Once introduced into a host cell, the vector produces the tagged protein. The purification involves equilibration, binding, washing, and elution steps, with careful consideration of buffer compatibility to maintain protein stability. The technique's advantages include its ability to isolate a specific protein from a mixture and its straightforward workflow. Post-purification, verification is done through SDS-PAGE and western blot.

05:01

📈 Advantages and Considerations of His Tag Purification

The second paragraph delves into the benefits of His tag protein purification, emphasizing its compatibility with cell culture media and lysates, ease of use, sensitivity, and specificity. It is noted as a cost-effective alternative to other chromatographic techniques such as gel filtration or HPLC. However, the method may require optimization to reduce non-specific binding, which involves adjusting the ionic strength of the equilibration and binding buffers. The paragraph also invites viewers to engage with educational content on the creator's social media platforms, including flashcards, notes, and daily MCQs with prizes, and provides information on how to support the channel through Patreon, the Beam Upi app, or the Super Thanks feature.

Keywords

💡His-tag protein purification

His-tag protein purification is a technique used to purify proteins with a single step affinity chromatography. It involves the use of a nickel-nitrilotriacetic acid (Ni-NTA) matrix that binds specifically to a hexahistidine tag attached to the protein of interest. The process is central to the video's theme, illustrating a method for separating and isolating specific proteins from a mixture. The script describes this process as having a specific advantage in identifying and purifying proteins within complex mixtures.

💡Affinity chromatography

Affinity chromatography is a type of chromatography that separates molecules based on their affinity to a particular ligand, in this case, the interaction between the Ni-NTA matrix and the hexahistidine tag. The video script explains that this technique is the key principle behind the separation in His-tag protein purification, allowing for the purification of proteins through a specific binding interaction.

💡Hexahistidine tag

A hexahistidine tag is a sequence of six consecutive histidine amino acids that is added to a protein to facilitate its purification using metal affinity chromatography. The script mentions that this tag is attached at the DNA level to the protein of interest, enabling it to bind specifically to the Ni-NTA matrix, which is crucial for the purification process.

💡Expression vector

An expression vector is a DNA molecule used to introduce a gene of interest into a host cell, where it can be expressed as a protein. In the context of the video, the expression vector is used to clone the open reading frame of the protein and attach the hexahistidine tag, which is essential for the subsequent purification process.

💡Equilibration buffer

The equilibration buffer is used in the first step of the purification process to prepare the column material for the specific pH and ionic conditions needed for the protein of interest. The script emphasizes the importance of this buffer in ensuring compatibility with the protein to maintain its stability and native state during purification.

💡Binding

Binding in the context of the video refers to the step where the protein of interest, tagged with hexahistidine, binds to the Ni-NTA matrix due to the specific coordinate bonds between them. This step is critical as it allows the selective capture of the tagged protein from a mixture of other proteins.

💡Wash buffer

A wash buffer is used in the purification process to remove any non-specifically bound proteins or contaminants from the column. The script describes this step as essential for ensuring that only the specifically bound protein of interest remains attached to the matrix, thereby increasing the purity of the final product.

💡Elution

Elution is the final step in the purification process where the specifically bound protein is released from the matrix. The script explains that an altered pH and ionic strength in the elution buffer are used to loosen the binding between the matrix and the protein, allowing the protein to be collected.

💡SDS-PAGE

SDS-PAGE, or sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used to separate proteins based on their molecular weight. The video script mentions using SDS-PAGE to verify the success of the purification process by checking the presence and purity of the protein of interest.

💡Western blot

Western blot is an analytical technique used to detect specific proteins in a sample. The script suggests using Western blot as a method to further confirm the presence of the purified protein, adding an additional layer of verification to the purification process.

💡Optimization

Optimization in the context of the video refers to the process of adjusting parameters such as ionic strength and pH to minimize non-specific binding during the purification process. The script notes that while this technique is specific and important, it often requires some standardization to ensure the best results.

Highlights

His tag protein purification is a single-step affinity chromatography method using immobilized metal ion chromatography.

Proteins are tagged with hexahistidine for specific interaction with the nickel NTA matrix.

The hexahistidine tag allows for the separation of the protein of interest from a mixture of proteins.

The expression vector is used to attach the His tag at the DNA level, either N-terminal or C-terminal.

The vector introduction into bacterial or mammalian cells leads to the production of tagged proteins.

The workflow of His tag purification includes equilibration, binding, washing, and elution steps.

Equilibration ensures the column material is soaked and compatible with the protein of interest's pH.

Binding involves the interaction of the tagged protein with the matrix via non-covalent coordinate bonds.

Washing removes non-specifically bound proteins, retaining only the specific interactions.

Elution uses a buffer with altered pH and ionic strength to release the bound protein.

SDS-PAGE and Western blot can be used to verify the success of the purification process.

His tag purification is compatible with cell culture media and lysates, easy to use, sensitive, and specific.

It is a cost-effective alternative to other chromatographic techniques like gel filtration or HPLC.

Optimization may be required to minimize non-specific binding and standardize buffer conditions.

The technique's specificity is a significant advantage for protein purification.

Follow the instructor on social media for flashcards, notes, and daily MCQs on related topics.

Support the channel through Patreon, BeamUpi app, or by clicking the Super Thanks option.

Transcripts

in this video we'll talk about

his tag protein purification

his tag protein purification is a method

by which proteins can be purified by a

single step affinity chromatography by

using immobilized metal ion

chromatography now in this case

the affinity is the key principle behind

separation

in this technique a ni ntm matrix is

used a nickel nta matrix is used

and the protein is tagged with

hexahistidine

and there is an interaction between

these matrix and this particular

histidine tag which is very specific and

there are non-covalent interactions like

coordinate bonds between these two

and that that is how the protein gets

attached with this matrix and it's easy

to separate

so where does the histac come from

actually we have to attach this his tag

with the protein

in order to do so we have to start with

an expression vector where we clone our

open reading frame

and

in the n-terminal or the c-terminal of

this particular protein one can attach a

hexa-histidine tag at the dna level

now once this particular vector is

introduced into the bacterial culture or

let's say mammalian cell which depends

on the type of expression vector we are

using

these expression vector would give rise

to the protein inside these cells

and ultimately these proteins would be

tagged with this hexahistidine

so what is the advantage of this kind of

tag

using this tag we we can understand

where is our protein of interest in a

mixture of other proteins so we can

separate them from a mixture of proteins

now like any column chromatography this

particular

chromatography has a simple workflow

so it starts with a equilibration step

then a binding step where our protein of

interest binds

then a wash and a illusion step

let us go through these steps

in a bit slowly so first step is

equilibration where we run our

equilibration buffer through the column

in this equilibration step the column

material gets soaked and equilibrated

for a particular ph

now the buffer that we have are using it

has to be compatible with the protein of

interest that we want to purify

factors to keep in mind while choosing

this buffer are ionic strength and ph

so factors such as ionic strains are

limited by protein stability so if we

change the ionic strength the protein

might get denatured and while we are

purifying the protein we don't want a

denatured protein right we want it in

its native state

next step is binding here we give the

protein

or the cell lysate

so in this step

there are multiple proteins which are

present in the lysate

but our protein is tagged so it is able

to bind to the matrix now protein

samples are generally dissolved in

specific buffers and loaded into the

columns proteins bind to the column via

several non-covalent interaction in this

case

the non-covalent interaction is

coordinate bonds

once protein have been immobilized in

the stationary phase then we have to use

wash buffer so the next step is washing

washing nco ensures that you don't have

any other non specific bound stuff in

the column all the non-specific untagged

proteins would be washed away in this

stage during the stage of washing all

the

all the weak bonds or non-covalent

interactions are broken down and only

the specific interactions are retained

now last step is illusion in this

illusion step we want to collect the

protein that is bound in the column

so

after all the non-specific interactions

are removed now we are using a

particular buffer which has a altered ph

and ionic strength which would loosen up

the binding between the matrix and the

protein of interest and then it would

come down into the

tube and we can collect that

so now we understand how we can use his

tag protein purification to purify our

protein of interest from a mixture of

protein

once we have purified our protein it is

important to

understand and cross check whether we

have uh whether this assay has worked or

not in order to do that we can run an

sds page and further do a western blot

against this particular protein

anyway let's talk about the benefits of

this purification this is compatible

with cell culture media and lysates

easy to use and quite sensitive and

specific

it is cheaper than many other

chromatographic technique such as gel

filtration chromatography or let's say

other kind of hplc techniques

anyway there are certain disadvantages

but that can be overcome the

disadvantage includes

this technique often requires

optimization to minimize non-specific

binding so many of the cases one has to

calibrate the ionic strength of the

equilibriation buffer or let's say the

binding buffer etc so little bit of

standardization is required but this

technique is really really specific and

important

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