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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

Vectors
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.
References
lk Process guide - Expression system - E.coli
http://www.abrf.org/ResearchGroups/ProteinExpressionResearchGroup/Activities/Page-PERG2007.pdf
http://www.mnstate.edu/provost/Recombexpresguide.pdf
http://www.cardiff.ac.uk/biosi/staffinfo/ehrmann/tools/vectors.htm
http://bitesizebio.com/articles/choosing-a-competent-ecoli-strain/
http://www.embl.de/pepcore/pepcore_services/cloning/choice_vector/ecoli/vectorfeatures/index.html