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Wednesday, September 21, 2011

Recombinant Protein Expression In E.coli

Procaryotic systems are well studied and widely used for protein expression, especially E.coli, The major advantages of E.coli system is
  1. Simple, well-understood genetics.
  2. Its very easy to genetically manipulate.
  3. Culturing cost is minimal.
  4. Expression is fast, since the doubling time is only 20 - 30 mins.
  5. Well established labeling protocols for stability studies.
  6. Established regulatory track record.
  7. Fermentation, easy to scale up.
  8. Inclusion bodies may be easy to purify.
  9. No unintended glycosylation.
  10. No viral or prion contamination risk.
Due to these advantages, it is preferred as an ideal system for protein expression, some of  the examples for recombinant proteins expressed in E.coli are Insulin, Taq Polymerase, etc.

The E. coli expression system has a proven track record over more than 30 years. It was the first expression system introduced in the biotech industry (recombinant insulin) and gram negative bacteria is still used for expression of a wide range of biopharmaceutical products. E. coli offers fast proliferation and the fermentation period is a few days only, offering high utilization degree of the upstream facility. The expression level is high, 1-5 g/L cell culture (intra cellular expression) often resulting in formation of insoluble inclusion bodies. The intra cellular protein is typically expressed with an extra N-terminal methionyl residue, which may have immunogenic consequences. 

Strains of E.coli
Depending on the nature of work, there are various types of strains which is specifically good for that particular type of work. So choosing the type of strain is the first thing before starting up the experimentation part. Few of the E.coli strain and applications are

Strain Application
XLI-Blue, DH5-alpha, top10 : Routine Cloning/Sub-cloning, Blue/white screening
XL10-Gold, MegaX DH10b : Very high efficiency cloning e.g. for library construction
XL1-Blue MR : Cloning of unmethylated DNA
JM110, INV110 : Production of unmethylated DNA
Sure, Stbl4 : Cloning of unstable plasmids
BL21 (DE3) : Expression from T7 promoter
BL21 (DE3) pLysS : Expression from T7 promoter, tight regulation
BL21 codon plus, Rosetta : Expression from T7 promoter with codon bias correction
Origami : Improved disulphide bond formation
Mach1 : Fast cloning (due to quick cell growth)
few strain and applications are listed here to find complete list click here

Vector is a DNA molecule used as a vehicle to transfer foreign genetic material into the cell. The four major types of vectors, they are plasmids, viruses, cosmids, and artificial chromosomes.
Here are the basic features of an E.coli expression vector which is needed for the cloning and expression of recombinat protein
Vector should have the following features
  • Selectable marker : Anitibiotic resistant genes used for Screening
  • Regulatory gene (repressor)
  • Origin of replication
  • Promoter
  • Transcription terminator
  • Shine-Delgarno sequence
  • Start codon and Stop codon
  • Tags and fusion proteins
  • Protease cleavage site
  • Multiple cloning site
pUC19 vector for Cloning
pUC 19 Vector - Image Source

There are many vectors commercially available for various kinds of protein expression and stability studies, strains and vectors can be obtained from Invitrogen or promega, there are so many other manufacturers also provides the strain and vectors.
Some of the examples of E.coli vectors are
pET series from Novagen
pALTER from Promega
pCal-n from Stratagene
pBAD/His from Invitrogen, etc
To see in detail click here or visit respective manufacturers website.

Cloning kit will have strain, plasmid and other reagents to carryout the experiments, vector (plasmid) provided in the kit will be minimal so this need to be amplified, this can be done by transferring the plasmid into suitable host strain(Transformation) and culture it in a small conical flask, overnight culturing will be enough to get lot of plasmid, plasmid isolation/ Plasmid extraction has to be done from the overnight culture and the pure plasmid is isolated and stored for long time in -20o C. Before plasmid extraction few ml of the culture can be taken for Glycerol stock preparation usually 15% Glycerol stock is prepared and can be stored in -70oC, this is useful because you dont have to do transformation every time, instead whenever plasmid is required a vial of glycerol stock can be inoculated on to the media and plasmid extraction can be done.

Once you have enough plasmid in hand, cloning work can be started, plasmid can be cut open using restriction enzyme which cuts at the specific region, cloning kit manual will provide the details on restriction enzymes and region where it cuts in the plasmid. If only one enzyme is used for the restriction digestion then it is called single digestion and if two restriction enzymes are used then its called double digestion. Depending on the sequence to be cloned and which site its need to be cloned, single or double digestion is chosen.

Once the plasmid is digested (Cut), foreign DNA (Target) can be ligated into it using enzyme DNA ligase usually that reaction is called as Ligation reaction.The digested and ligated products can be run on agarose gel to check whether there is any undesired product formation by analyzing the size of the product on gel. once the ligation is done this need to be transferred to the host cell (E.coli), either transformation or electroporation methods can be used for transferring. Transformation is mostly used, to make the cells take up DNA, bacterial cells need to be chemically treated usually cacl2 is used which enhances the foreign DNA uptake and the cells which has the ability to take up foreign DNA is called Competent Cells. Transformation works by by giving a heat shock (42oC for 2 mins) to the bacterial cells and then reducing the temperature (on ice for 5 mins). Procedure involves mixing up of bacterial cells and the foreign DNA and keeping it in ice for 30 mins then a heat shock at 42oC for 2 mins incubate in ice for 5mins then add few ul of sterile culture media (LB) and incubate at 37oC for 1hr, this step will help bacterial cells to recover from the heat shock, the culture can be plated on LB Agar with suitable con. of antibiotic, suitable screening method depending on the plasmid can be used, select different colonies and shake flask studies can be done.

Shake Flask Studies and AnalysisUsually the selected colonies are inoculated into small screw cap tube with 5-10 ml media and appropriate con. of antibiotics, culture is grown to a particular OD 0.6 – 0.8 then the culture is induced with an inducer. IPTG acts an inducer, the princeiple involved in Induction using IPTG is, Expression of many proteins in bacteria is controlled by the Lac Operon. An operon is a collection of linked genes under common, coordinate control. Typically bacteria do not use lactose as a source for food, however when enough lactose is added to the cells, lactose binds to repressor proteins and will cause the induction of the production of two different proteins, permease, used to transport carbohydrate and ß-galactosidase. ß-galactosidase will hydrolyze the disaccharide lactose to the monosaccharides glucose and galactose, then the bacteria can continue to grow. Many researchers and companies have removed these two genes and placed a gene for a protein that they want. This way, when lactose or its non-metabolized inducer isopropyl beta-thiogalactoside, (IPTG), is added the cell is sort of tricked into making the protein cloned into the lac operon rather than permease and ß-galactosidase.
Induced samples were taken at regular intervals (0hr, 6hr, 12hr, 24hr, etc) and can be analyzed on SDS-PAGE gel, depending on the expressed protein gel percentage can be made. An uninduced sample and a protein marker of suitable range is also loaded. The clone which produces high amount of protein is selected and used for scale up. Biochemical Protein Assays can be done to quantify the expressed protein. Protein characterization need to be done for the expressed protein which will help in Downstream and protein purification Steps.
lk Process guide - Expression system - E.coli

Wednesday, September 14, 2011

Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC)

Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC)

Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC)

Gel filtration chromatography (GF) (also known as molecular exclusion chromatography) relies on a stationary phase consisting of spherical gel particles whose size and porosity are carefully controlled during manufacture.

The alternative names for Gel filtration chromatography are molecular sieve chromatography, gel permeation chromatography.

The main application of gel filtration chromatography / size exclusion chromatography is desalting, removal of salts from the sample.
When the mobile phase is passed through a column of this material, molecules are fractionated on the basis of their sizes and shapes. Small molecules are able to diffuse into the pores, while larger molecules are excluded. This occurs repeatedly as the mobile phase moves down the column, and as a result, during the elution process, the small molecules are retarded with respect to the larger molecules. This causes the smaller molecules to appear in the later fractions of the eluate.
Size is not the only factor that influences the rate at which solutes are retarded. For a given pore size, rod-like molecules will tend to be excluded with respect to spherical molecules of the same size. In addition, certain molecules may have some affinity for the stationary phase as a result of non-covalent interactions.

gel filtration chromatography, size exclusion chromatography

The solid support used in gel filtration chromatography is called media or matrix. it is available in different ranges depending upon the nature of impurity need to be removed. GE lifescience has the gel filtration matrix in the name of Sephadex G-10, Sephadex G-25Sephadex G-50Sephadex G-75 etc
G-25 has the molecular sieving ability of 1KDa - 5KDa and the chromatography matrix is available as G-25 coarse,G-25 medium and G-25 fine the classification is based on the particles.

Bio rad has gel filtration matrix in the name of Biogel P10, Biogel P4, Biogel P2, Biogel P30 etc, it is polyacrylamide based, the number denotes the molecular cut off Biogel P4 has molecular cut off range of 800 - 4000 Daltons(0.8 - 4 KDa)

Storage of Gel Filtration Matrix
If the chromatography matrix is not using for long time it can be stored at 4 degree Celsius with 20% Ethanol. while reusing after storage ethanol should be removed by repeated washing in distilled water.

Advantages / Applications of Gel Filtration Chromatography (GF) / Size Exclusion Chromatography (SEC):
  • Separation process is carried out under very mild conditions, from 37 ºC. to cold room conditions.
  • High resolution can be achieved.
  • A wide range of buffer systems may be used (buffer molecules and ions are generally excluded).
  • Small molecules or ions that stabilize the biomolecules may be added to the buffer.
  • Separation process is independent of ionic strength, so elution is carried out isocratically(only one buffer is used).
  • Gel filtration can be used for desalting and/or buffer exchange.
  • Gel filtration can be used to obtain an estimate of molecular size.
  • A wide range of porous gels are available commercially.
Precautions to be taken while using Gel Filtration chromatography Matrix
Since the fractionation or separation is based on the pore size of the particle (Matrix) damage in the particle can affect the separation process. 
Buffers and matrix should be degassed, air bubbles entering the column can lead to poor resolution.

Ion Exchange Chromatography Principle

Ion Exchange Chromatography is of two types Cation Exchange chromatography and Anion Exchange Chromatography
Cation Exchange Matrix will have -vely charged particles, Anion Exchange Matrix will have +vely charged particles

At isoelectric point (pI) net charge of the protein will be zero. when the pH of the protein is reduced below its pI it becomes positively charged, and if the pH of protein is raised above its pI value it becomes negatively charged.

MechanismTo optimize binding of all charged molecules, the mobile phase is generally a low to medium conductivity (i.e., low to medium salt concentration) solution.The adsorption of the molecules to the solid support is driven by the ionic interaction between the oppositely charged ionic groups in the sample molecule and in the functional ligand on the support. The strength of the interaction is determined by the number and location of the charges on the molecule and on the functional group.By increasing the salt concentration (generally by using a linear salt gradient) the molecules with the weakest ionic interactions start to elute from the column first. Molecules that have a stronger ionic interaction require a higher salt concentration and elute later in the gradient. The binding capacities of ion exchange resins are generally quite high. This is of major importance in process scale chromatography, but is not critical for analytical scale separations.

Ion Exchange Chromatography Principle - Image Source

Separation of molecules by ion-exchange (IEX) chromatography relies on differences between the net surface charges on the solute molecules. Proteins, for example, contain numerous groups which can ionize to varying extents depending on the pH of the solution. The ionic state of these groups is highly dependent on the pH, and as a result, the net surface charge of a protein will undergo a change as the pH of their environment varies.
At the isoelectric point (pI) of the protein, the protein will have little or no tendency to bind either to a cationic stationary phase (that is, one which has positively charged groups) or to an anionic stationary phase (one that has negatively charged groups).
At pH value below the pI, the protein will have a net positive charge, and will tend to bind reversibly with to the surface of a cation-exchange resin, that is, one that has negatively charged groups at that pH.
Note that a cation-exchange resin is anionic, having negatively charged groups, while an anion exchange resin is cationic, since it has positively charged groups.
Binding to the matrix requires that buffer ions that are bound to the matrix during the equilibration process be displaced by the solutes of interest. Thus, and exchange of ions takes place at the surface of the matrix.
On applying the sample, conditions are chosen so that as many as possible of the unwanted solutes do not bind to the resin, leaving the molecules of interest bound to the top of the column. When all the unwanted solutes have been eluted, the composition of the buffer is gradually altered, either by increasing the ionic strength, Γ , or, less commonly, by changing the pH of the eluting buffer. This process is called gradient elution. The bound solutes are eluted at different values of the ionic strength, as buffer ions compete with the bound molecule for cationic or anionic sites on the resin.

You might also be interested in Protein Purification: Principles, High Resolution Methods, and Applications by Jan-Christer Janson, Wiley Publishers, Leading experts in the field cover all major biochemical separation methods for proteins in use today, providing professionals in biochemistry, organic chemistry, and analytical chemistry with quick access to the latest techniques

Matrix Materials
here is a variety of commercially available materials which can be classified as follows:
Anion exchangers Type Functional group
Quaternary ammonium (Q) Strong -OCH2N+(CH3)3
Diethylaminoethyl (DEAE) Weak -OCH2CH2NH+(CH2CH3)2
Diethylaminopropyl (ANX) Weak -OCH2CHOHCH2NH+(CH2CH3)2

Cation exchangers Type Functional group
Sulfopropyl (SP) Strong O-CH2CHOHCH2OCH2CH2CH2SO3
Methyl sulfonate (S) Strong O-CH2CHOHCH2OCH2CHOHCH2SO3
Carboxymethyl (CM) Weak O-CH2COO
Strong ion-exchangers do not show any marked change in their ionic states with changes in pH. They remain fully charged over a broad range of pH values.

Advantages of strong ion-exchangers:

  • the development and optimization of separations is fast and easy since the charge characteristics of the medium do not change with pH;
  • the mechanism of interaction is simple since there are no intermediate forms of charge interaction;
  • sample loading (binding) capacity is maintained at high or low pH since there is no loss of charge from the ion exchanger.
Most proteins can be separated on either strong or weak ion-exchangers. If strong ion-exchangers do not give the required results, try a weak exchanger. These offer a different seletcivity to strong exchangers, but their exchange capacity changes with pH.
If the elution process is carried out using the same buffer that was used in applying the sample, the elution is said to be ISOCRATIC. Alternately, the elution may be carried out for a while with that buffer, and then with another buffer. This is called STEP-WISE ELUTION. For the best results, the composition of the eluting buffer is changed as the elution progresses. The change is usually a change in ionic strength, from a less dilute to a more concentrated buffer, or, less frequently, a change in pH. This elution process is called GRADIENT ELUTION, and this generally gives a much better separation of solute.

Hydrophobic Interaction Chromatography Principle

Salting Out:
There are hydrophobic amino acids and hydrophilic amino acids in protein molecules. After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of solvation and allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water.
When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available to interact with the charged part of the protein. As a result of the increased demand for solvent molecules, the protein-protein interactions are stronger than the solvent-solute interactions; the protein molecules coagulate by forming hydrophobic interactions with each other. This process is known as salting out.
HIC Mechanism
Separation Based on Hydrophobic Interaction
The hydrophobic ligands on HIC media can interact with the hydrophobic surfaces of proteins. In pure
water any hydrophobic effect is too weak to cause interaction between ligand and proteins or between
the proteins themselves. However, certain salts enhance hydrophobic interactions, and adding such salts brings about binding (adsorption) to HIC media. For selective elution (desorption), the salt concentration is lowered gradually and the sample components elute in order of hydrophobicity.
The final result of a HIC separation is based therefore on interplay between the prevalence and
distribution of surface-exposed hydrophobic amino acid residues, the hydrophobicity of the medium
the nature and composition of the sample, and the type and concentration of salt used in the buffers.

HIC media are composed of ligands containing alkyl or aryl groups coupled to an inert matrix of
spherical particles. The matrix is porous, in order to provide a high internal surface area, while the ligand plays a significant role in the final hydrophobicity of the medium.

Sodium, potassium or ammonium sulfates produce relatively high precipitation. It is these salts
that effectively promote hydrophobic interaction and have a stabilizing influence on protein
structure. In practice sodium, potassium or ammonium sulfates effectively promote ligand-
protein interactions in HIC and have a stabilizing influence on protein structure. Hence the most
commonly used salts are (NH4)2SO4, Na2SO4, NaCl, KCl and CH3COONH4.
If the protein of interest does not bind under high salt conditions, use a more hydrophobic
medium. If the protein of interest binds so strongly that non-polar additives are required for
elution, decrease the salt concentration in the start buffer or use a less hydrophobic medium.