2 Analysis of Recombinant Proteinsweb.xidian.edu.cn/yqxia/files/20140222_213148.pdf · Outline 3 Protein structure Protein folding Protein stability Analytical techniques u riR6 o]åz

By Yuqiong Xia2014

Analysis of Recombinant Proteins

Pharmaceutical Biotechnology

Keywords

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Amino acid, α-helix, β-sheet, β-turn

Protein folding, refolding, denatured

Fluorescence, oligmeric, oligo-

Blotting, gel electrophoresis, chromatography

生物制药工程夏玉琼西安电子科技大学

Outline

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Protein structure Protein folding Protein stability Analytical techniques


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Amino acid生物制药工程夏玉琼西安电子科技大学

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Primary structure生物制药工程夏玉琼西安电子科技大学

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Nonpolar amino acids生物制药工程夏玉琼西安电子科技大学

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Charged amino acids生物制药工程夏玉琼西安电子科技大学

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Polar生物制药工程夏玉琼西安电子科技大学

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G-CSF （granulocyte-colony stimulating factor）生物制药工程夏玉琼西安电子科技大学

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Secondary structures

http://quizlet.com/13505003/amino-acids-carbohydrates-lipids-structure-and-trivia-flash-cards/


α-helix

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Peptide NH and C=O groups have electric dipole moments Positive partial charge at N terminal and negative at C terminal


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Helical wheel of Erythropoietin

plot every 360o/3.6 = 100o around a circle


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β-sheet生物制药工程夏玉琼西安电子科技大学

β-turn and loop

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β-turn Proline and glycine

Short hairpin loops Link β-strands

Long loops hydrophilic

http://ww2.chemistry.gatech.edu/~lw26/structure/protein/secondary_structure/beta_turn/index.html


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Structure of G-SCF: predicted by software

alpha

betaturncoil


Structure of G-SCF: predicted by software

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What impacts on protein folding The tendency of each

amino acid to form certain secondary structure

the hydrophobicity of their side chains


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Tertiary and Quaternary structures

http://www.umass.edu/molvis/workshop/imgs/protein-structure2.png


Outline

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Why protein refolding

http://web30.webbox300.server-home.net/789001/Uploaded/scil%7CProt_Fold_Protocols.jpg

Recombinant proteins are always aggregates!


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How to refold proteins

http://2012books.lardbucket.org/books/introduction-to-chemistry-general-organic-and-biological/section_21/9e024c11709a6bb80a04338d399c3a20.jpg

1

2

1. Use detergent to dissolve proteins

2. Remove detergent


How to refold proteins: for S-S

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Air oxidation Add glutathione (谷胱甘肽) (catalyst) Reduction and oxidation S-S reshuffling (重组)


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Refolding processes

http://www.tsinghua.edu.cn/publish/zhpyen/1181/20101212175322899680164/protein7.jpg


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How to refold more efficiently

http://www.nature.com/nature/journal/v487/n7407/full/487308a.html?WT.ec_id=NATURE-20120719http://upload.wikimedia.org/wikipedia/commons/4/42/Polyethylene_glycol.pnghttp://www.topsan.org/Proteins/JCSG/3g7p

Use chaperone(分子伴侣)

Use polyethylene glycol (PEG)


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Circular Dichroism

http://www.proteinchemist.com/cd/cd7.gif

α-helix 208 nm 222 nmβ-sheet 218 nm


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Fluorescent spectrum

http://www.rsc.org/ej/PP/2008/b804450n/b804450n-f5.gif


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Fourier transform infrared spectroscopies (FTIR)

http://www.shimadzu.com/an/industry/pharmaceuticallifescience/proteome0205005.htmhttp://www.lwgzs.com/read.php?tid=24152

β-sheet 1610-1640 cm-1

Random 1640-1650 cm-1

α-helix 1650-1658 cm-1

β-turn 1660-1700 cm-1

Bovine serum albumin


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X-ray crystallography

http://users.ba.cnr.it/ic/crisrc25/Protein_Crystallography.html


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How to know if protein is folded

T/oC

F FoldedUnfolded

Fluorescence

Unfolded

Folded

DSC

T/oC

ΔH

Folded

Unfolded

Inte

nsity

Sedimentation coefficient

Sedimentation velocity Unfolded

Folded

Inte

nsity

t/min

SEC/GPC


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How to determine if the protein is oligomeric

http://www.nanolytics.de/index.php?lg=en&main=biochemistry

dimer

monomer

Marker

“Gold standard”


Outline

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Chemical stability of proteinHydrolysis (CO-NH) Oxidation


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Physical stability of proteinNatural Denatured Denatured Aggregates

Lyophilize (freeze-dry) to increase the stability

Modified from http://2012books.lardbucket.org/books/introduction-to-chemistry-general-organic-and-biological/section_21/9e024c11709a6bb80a04338d399c3a20.jpghttp://www.baxterbiopharmasolutions.com/sterile-manufacturing-solutions/traditional-manufacturing/lyophilized-vials.html


Outline

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Blotting Techniques

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Western blotting Transfer proteins from gel to membrane

Emembrane

gel


Blotting Techniques

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Detect Ag on membrane

Ag antigen

Ab antibodyB biotin

SA streptavidin

E enzyme

S substrate

P product


ELISA(酶联免疫吸附法)

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Sandwich Ab-Ag-Ab


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Gel electrophoresis

https://ww2.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/page_protein.html

SDS-PAGE Native gel

electrophoresis Blue native

electrophoresis


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2D Gel electrophoresis

http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/Two_Dimensional_Polyacrylamide_Gel_Electrophoresis(2D-PAGE)


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Capillary Electrophoresis

http://theses.ulaval.ca/archimede/fichiers/24330/apa.html


Chromatography

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Measure the amount and purity of protein


Size Exclusion Chromatography

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Separates proteins based on their size or molecular weight or shape

native protein

Aggregated

Degraded


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Ion Exchange Chromatography

http://www.reachdevices.com/Protein/ProteinPurification.html

Cation-Exchange Chromatography


Bioassay

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Detect the bio-function of a protein

http://info2.gbiosciences.com/complete-bioassay-handbook

3H-T was used as probe


Mass spectrometry

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Measure molecular weight of protein

Singly charged

doubly charged


Summary

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Protein primary and secondary structure Protein refolding Analytic techniques: ELISA, ionic exchange

chromatography, 2D gel electrophoresis


Homework I

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Can we perform the 2D electrophoresis by doing SDS-PAGE first and then IEF? Why?

Now we have a mixture of three proteins. They carry different amount of negative charges: -10 for protein A, -20 for protein B, and -30 for protein C. Please design a method to separate them.


Documents

2 Analysis of Recombinant Proteinsweb.xidian.edu.cn/yqxia/files/20140222_213148.pdf · Outline 3 Protein structure Protein folding Protein stability Analytical techniques u riR6 o]åz