SCT60103 Gene & Tissue Culture Technology

Technology used for High Level

Expression and Purification of

Recombinant ProteinsChow Sook Yee (0328298)

Clayton Voon (0327187)Saseedaran (0327564)

Ong Ze Liang (0327055)

INTRODUCTION

a) In vivo

• cell based protein expressiontransfecting cells with DNA vector,then culturedcells are lysed and protein will extracted

• Exp: yeast cell- the efficient production of fermented beverages, like beer, wine and cider as well as bio-ethanol

b) In vitro

• cell free protein expression

• whole cell extract with the component for translation

• Exp: rabbit reticulocytes (RRL) synthesis of virus-like particles for vaccines and drug therapy

What is Protein Expression and Purification- study of specific protein regulate biology by producing functional protein of interest

IntroductionSteps for a in vivo cell based system:

• Transfection

• Lysing protein with tags

• Purification of protein

• Functional array

Bacterial protein expression systems Mammalian in vivo expression

• Popular option because bacteria are easy to culture, grow fast and produce high yields of

• recombinant protein.

• This expression systems usually produce functional protein

• Multi-domain eukaryotic proteins expressed in bacteria often are non-functional

• Protein yield is low, cost of production is high and time-consuming.

• Not conducive to either high throughput protein synthesis or expression of proteins that are toxic to host cells.

The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield

Transfection• Process of inserting genetic material into a eukaryotic cell

• Expression or production of proteins using cell own machinery

• Several pHUE were constructed using different number of genes

• Gene obtained were from previously plasmid containing cDNA clone.

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3

2

EcoRI, ClaI, and HindIII sites were digested by EcoRI/HindIII followed by blunt ending with Klenow and self-ligationFurther digested by BamHI and double-stranded oligonucleotide

This results in pET15b.ep and is then digested with NdeI and BamHI

Ligation of DNA encoding a Ub ORF from UBA52 cDNA

clone .

• Isopropyl-1-thio-β-D-galactopyranoside (IPTG) is added into E.coli strain BL21 (DE3) for protein expression.

• T7 promoter is used for RNA polymerase to transcribe mRNA to make necessary proteins

• Different pHUE vectors were analyzed using SDS- page

• Synthesis of Ub fusion protein detected by abundant blue appears in SDS- page.

• Reflects high level of protein expression

• Further expression by sequencing N-terminal with Edman degradation

• Result in precise match without DUB present

• Indicates Ub fusion being accurately cleaved in E.coli.

Expression of recombinant protein

• commercial vectors use a T7 promoter

• The lac repressor (LacI) binds in a sequence specific manner to the major groove of the operator sequence and blocks T7 RNA polymerase from binding the promoter sequence.)

• The GST Tag

Glutathione S-transferase (GST) is a 211 amino acid protein (26kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins.

Protein affinity tagsProtein affinity tags – Expression vectors used for protein production usually

contain a specific tag for affinity purification

• The poly His tag

The DNA sequence specifying a string of six

to nine histidine residues is frequently used

in vectors for production of recombinant

proteins.

Most Purification tags coupled with protease cleavage sites to remove the purification tag and simultaneously elute the proteins

• A major drawback of this approach is the covalent linkage of the two proteins, where the presence of the fusion partner may prevent or interfere with subsequent use of the desired protein

• overcome this problem a protease recognition site can be constructed between the two fused proteins; however, this involves altering the N terminus of the desired product, resulting in the expression of an unauthentic protein

Fusion partner

ubiquitin (Ub). This small eukaryotic protein provides two benefits.

• it offers a natural yield enhancement

• fusion to Ub generally leads to increased protein solubility

Fusion tags

• The cells are frozen at −70°C after the transfection process

• In order to obtain the produced proteins the cells are lysed using a sonication system

• 3 × 1 min bursts at 0°C)

• Soluble protein fraction recovered by centrifugation at 4°C (15 min at 15,300g)

Sonification cell lysis

• IMAC is based on the interaction of proteins with certain amino acid residues on their surface and divalent metal ions (e.g., Ni2+, Cu2+, Zn2+, Co2+) immobilized via a chelating ligand.

Column is washed using the 50 mM Na2HPO4/NaH2PO4 (pH 8.0), 300 mM NaCl

Equilibration of the column with a binding buffer containing a low concentration of imidazole

Proteins with histidines bind the column while displacing the imidazole counter ligands

Elution of bound proteins is performed using a gradient of imidazole up to 100 to 500 mM

Purification using IMAC

Conclusion• We can understand the fundamental of protein expression which is the 3 simple

steps

• The limitations of this finding is although DUBs that have been isolated from various species they are relatively large enzymes and difficulties have been encountered with expressing and purifying large quantities, and in finding a stable DUB and cleavage efficiency of our chosen DUB against a Ub-proline bond, which has been an observed limitation of most ubiquitin-specific proteases

• Based on the finding, we can conclude that protein recombination with ubiquitin will delivers high-level expression, includes an easy means of purification, and allows the production of a variety of proteins , peptides with authentic N termini for a range of downstream applications, meeting many of the demands of heterologous gene expression with minimal experimental steps in a convenient, low-cost manner, and would be readily amenable to high-throughput applications

• The relatively small size of ubiquitin also makes this system attractive for metabolic labeling of proteins for applications such as NMR.

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