Discuss ONE(1) technology used for High Level Expression and Purification of Recombinant Proteins

High level expression and purification of recombinant proteins from Escherichia coli with HIS-TAGGROUP 8:

Tan Kai Li ( 0322005 )Lee Ling XIan (0326735)Vyvian Lee Yee Ting (0326962)Cheah Jin Lu (0326156)

Introduction:➔ Recombinant protein expression technology enables analysis of

gene regulation, protein structure and function.➔ Originated in the early 1990s when Human Genome Sequencing

was initiated.➔ Can be expressed using:

- Cell based protein expression: bacteria, mammalian cell system, etc.

- Cell-free protein expression.➔ Protein purification: separate desired proteins from all others.

- By protein affinity tags such as the hexa-histidine (6xHis), glutathione S-transferase (GST) and etc.

Method

Recombinant Protein Production

Protein Expression Protein Purification (Bacterial System) (Protein Affinity tags- His Tags)

Method- Step 1: Protein Expression● Isolation and amplification of the gene of interest out of a

vector and subcloning it into a bacterial expression vector.

● Verification of the authenticity of the subcloned gene by restriction enzyme digest and sequencing.

● Transformation of recombinant constructs into a high efficiency expression bacterial strain.

● Test for the expression of the recombinant protein by SDS-PAGE and/or western blot.

● Cells are then lysed to extract the expressed protein for subsequent purification.

Figure 1: Bacterial protein expression (amsbio n.d.)

Method- Step 2 Protein Purification Protein affinity tags (Histidine Tags)

➔ Expression vectors used for protein production

usually contain a specific tag for affinity purification.

➔ Binding of the tags to specific resins help to facilitate

purification and elution of the proteins of interest.

➔ Expressed His-tagged proteins can be purified and

detected easily because the string of histidine

residues binds to several types of immobilized metal

ions, including nickel, cobalt and copper, under

specific buffer conditions.

Figure 2: Histidine codon on DNA encode for peptide tag (Biology Animation Video 2016)

Method- Protein PurificationImmobilized metal affinity chromatography (IMAC)

- A column consisting of agarose bead which attached to nickel atom

- Within the column, His tag on protein have affinity bind to nickel atom

- The protein lack of tag will be washed out of the column

- A low pH solution causes Histidine residues become protonated and unable bind to the nickel on the bead.

- A solution with a molecules called imidazole competes with histidine for nickel binding

Figure 3 & 4: IMAC for purify the His-tag protein (Biology Animation Video 2016)

Principle (Protein expression)Protein expression can occur in different location of the cells:

1.Insoluble intracellular2.Soluble intracellular3.Periplasmic4.Extracellular

➔ Insoluble intracellular is preferred due to formation of inclusion bodies, great yields, high purity

➔ Inclusion bodies are insoluble aggregates of denatured or partly denatured protein that lack biological activity but often allow high expression levels of the recombinant protein. Figure 5: Types of protein expression

Principle of protein expression

Figure 6: Anatomy of expression vector (L.Rosano, 2014)

Principle of Protein Expression E.coli bacteria expression system (T7 system)

➔ A multiple cloning sites provides number of sites for restriction enzymes to cut the plasmid.

➔ Operator sites derived from lac operon control when the tagged protein is expressed.

➔ When enzyme digest plasmid, it creates blunt ends on DNA.

➔ If the DNA with protein-coding region is also blunt ended, it can be ligated into this plasmid of E.coli host cells (Siegel, 2016).

Principle of protein expression

➔ In the cells, repressor proteins bind to lac O, creating an obstruction for RNA polymerase.

➔ When an inducer (eg: IPTG) is added to the culture medium, inducer binds to the repressor proteins and allows RNA polymerase to pass through and transcribe DNA into RNA (Biology Animation Video 2016).

Figure 7: Example of T7 system (Siegel, 2016)

Principle of affinity tags for purification

➔ Histidine tag can be fused to either the N- or C-terminus of a protein.

➔ Histidine-tagged proteins have a high selective affinity for Ni2+ and several other metal ions that can be immobilized on chromatographic media.

➔ Protein containing a histidine tag will be selectively bound to metal-ion-charged media while the proteins lack of tags will be eluted out from the column.

➔ This can separate tagged proteins from the cellular material.

➔ Low pH solution causes untagged protein become protonated and unable to bind to Ni2+ metal ions.

Figure 8: Principle of Affinity tags (GE Healthcare Bio-Sciences AB, 2012)

Principle of affinity tags for purification (con’t)

➔ Imidazole is utilized as a competitive agent for elution of histidine-tagged proteins.

➔ Presence of surface-exposed histidine residues or other contaminants amino acids can lead to unwanted binding of untagged host cell proteins.

➔ Imidazole can be added in low concentrations in the sample and binding buffer in order to reduce the binding of contaminant proteins, and thus increase the final purity.

(GE Healthcare Bio-Sciences AB, 2012) Figure 9: Affinity chromatography separation method

Limitations (Protein expression)

➔ E.coli lacks some organelles found in eukaryotic cells therefore certain eukaryotic genes will not function.

➔ E.coli lacks post-translational machinery and molecular folding.

➔ Production of insoluble protein due to formation of inclusion bodies that interrupt the protein-refolding procedures.

➔ Codon bias leads to translation errors when a heterologous gene with codons which are rarely used in bacteria are not properly expressed in bacteria.

Improvement (Protein expression)

Method 1: Modify the factors influencing the formation of insoluble fraction through a stringent control of the cellular milieu

Method 2: Refold the protein from the inclusion bodies to avoid target protein modification

Method 3: Engineer the target protein to achieve soluble expression through fusion protein technology

Limitations and Improvement (Purification)

➔ Bacterial proteins that are able to bind to the His-Tag will co-eluted with the recombinant protein.

➔ This can be partially ruled out by increasing the stringency of washing.

➔ For example, by increasing volume of washing buffer or add a low concentration of imidazole.

➔ If His-Tag has been degraded, purification must be done at lower temperature (-4c) to reduce the purification.

Conclusion:

Recombinant protein expression and purification is frequently useful for both basic research studies and commercial applications. High-throughput protein expression and purification has begun to revolutionize the manner in which studies are conducted in various research fields.

References- Alex Siegel 2016, How does IPTG-induced gene expression work at a molecular level?, viewed 27 Oct 2017,

<https://www.quora.com/How-does-IPTG-induced-gene-expression-work-at-a-molecular-level>- Amrik Basi, Gicell Schaenzler etc 2015, Protein expression handbook, viewed 28 Oct 2017,

<https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-expression-handbook.pdf>- Amsbio n.d, Bacterial Protein Expression, viewed 29 Oct 2017,

<http://www.amsbio.com/bacterial-protein-expression-services.aspx>.- Biology Animation Video 2016, Protein Purification Animation - his tag protein purification, viewed 28 Oct 2017,

<https://www.youtube.com/watch?v=DfzB6BtSjBY&t=16s>.- Germán L. Rosano, Eduardo A. Ceccarelli 2014, Recombinant protein expression in Escherichia coli: Advances and

challenges, viewed 29 Oct 2017, <https://www.researchgate.net/publication/262608486_Recombinant_protein_expression_in_Escherichia_coli_Advances_and_challenges>

- Mol Cell Biochem 2008, Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies, viewed 28 Oct 2017, <https://www.ncbi.nlm.nih.gov/pubmed/17874175 >

- Front Microbiol. 2014, Recombinant protein expression in Escherichia coli: advances and challenges, viewed 27 Oct 2017, < https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029002/>

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