Molecular Protein Techniques
• Protein is derived from the Greek proteois,
meaning “of the first rank”
• Proteins most abundant among biological
molecules
• The building blocks of proteins are amino acids
(there are 20 naturally occurring amino acids)
• ~40 000 genes in the human genome, but > 300
000 proteins in the human proteome
• Difference between the number of genes and
proteins is due the fact that one gene is able
to give rise to more than one protein (splicing phenomena)
• Post-translational modifications change subcellular localisation, activity and stability
of many proteins (done by enzymes, attach a phosphate, ubiquitin, signalling
molecule)
• Proteins are made of motifs and domains which determine their cellular functions
Human Genome Project
46 chromosomes, 3 billion bp, ~40,000 genes (~20,000 annotated)
Transcriptome 40-100 000 mRNAs
Proteome 100 -400 000 proteins
Protein interactions >106interactions
Protein >108 post-translational modifications
modifications
The power of recombinant proteins
• Expression of proteins or their domains in large quantities, necessary for
biochemical and structural studies
• Change primary protein sequences by site-directed mutagenesis and to anyalyse the
function of generated mutants in vivo
• Investigate subcellular localisation, post-translational modificiations, binding
partners
• To study function of a protein of interest in cells, organs or organisms (knocin or
knockout studies)
Application of recombinant proteins
• Biochemical studies – study enzymatic properties and regulation, identify binding
partners etc.
• Production of polyclonal and monoclonal antibodies
• Structural studies: Crystallography and NMR analysis
• Cell biology: microinjection into mammalian cells
,• Biotechnology: large-scale production of growth factors and hormones (eg.
interferon, insulin etc.)
• Pharmaceutical industry: develop screening assays for drug discovery programs
• Clinical: production of vaccines, diagnostic kits
1. Expression, Purification, Analysis of Recombinant Proteins
• For a long time, isolating protein was difficult – had to purify directly from a
tissue source, complexity of intact tissues and organs required long series of
chromatography steps – often yielded only few milligrams of pure protein
• For structural and functional analyses – lots of protein needed
• Nowadays – proteins can be grown in cell culture through genetic engineering
techniques
Protein expression techniques/systems
Expression in Prokaryotes
Bacterial (Vectors for constitutive and inducible expression)
Expression in Eukaryotes
Yeast (vectors: autonomously replicating and genome integrating)
Insect cells (baculoviruses)
Mammalian cells (transient transfections, stable cell lines, adeno-and retroviruses)
Aspects of bacterial expression Aspects of eukaryotic systems
- In bacteria many recombinant - Lower levels of expression (in most
proteins are misfolded, insoluble or cases)
degraded - More expensive (expression medium)
- Numerous post-translational - More complex and takes longer to
modifications do not occur in establish (~2 months for stable cell
bacteria, such as phosphorylation, line)
glycosylation
- Difficult to purify proteins >60kDa
Purification
• First step involves breaking open cells to release contents cell homogenate
- Break cells with high-frequency sound
- Mild detergent
- Force cells through small hole using high pressure
- Shear cells between close-fitting rotating plunger and thick walls of a glass vessel
• Centrifugation techniques to obtain proteins
, Protein separation techniques
a) By size
- Gel electrophoresis
- Gel filtration chromatography
- Ultracentrifugation
- Dialysis
b) By charge
- Isoelectric focusing
- Ion exchange chromatography
c) By polarity
- Paper and reverse-phase chromatography
- Hydrophobic interaction chromatography
d) By specificity
- Affinity chromatography – commonly used
Types of Electrophoresis
a) Filter paper
electrophoresis
- Proteins easily
denatured due to
high absorbance of
filter paper
- Works for small
peptides or amino
acids
b) Thin layer
electrophoresis
(TLC)
- Chemically-modified
cellulose
c) Gel electrophoresis
- Starch gel
electrophoresis
- Agarose gel electrophoresis
- Polyacrylamide gel electrophoresis (PAGE)’
Native-PAGE SDS-PAGE
• Enzyme activities are retained after • SDS (sodium dodecyl sulphate) coats
• Protein is derived from the Greek proteois,
meaning “of the first rank”
• Proteins most abundant among biological
molecules
• The building blocks of proteins are amino acids
(there are 20 naturally occurring amino acids)
• ~40 000 genes in the human genome, but > 300
000 proteins in the human proteome
• Difference between the number of genes and
proteins is due the fact that one gene is able
to give rise to more than one protein (splicing phenomena)
• Post-translational modifications change subcellular localisation, activity and stability
of many proteins (done by enzymes, attach a phosphate, ubiquitin, signalling
molecule)
• Proteins are made of motifs and domains which determine their cellular functions
Human Genome Project
46 chromosomes, 3 billion bp, ~40,000 genes (~20,000 annotated)
Transcriptome 40-100 000 mRNAs
Proteome 100 -400 000 proteins
Protein interactions >106interactions
Protein >108 post-translational modifications
modifications
The power of recombinant proteins
• Expression of proteins or their domains in large quantities, necessary for
biochemical and structural studies
• Change primary protein sequences by site-directed mutagenesis and to anyalyse the
function of generated mutants in vivo
• Investigate subcellular localisation, post-translational modificiations, binding
partners
• To study function of a protein of interest in cells, organs or organisms (knocin or
knockout studies)
Application of recombinant proteins
• Biochemical studies – study enzymatic properties and regulation, identify binding
partners etc.
• Production of polyclonal and monoclonal antibodies
• Structural studies: Crystallography and NMR analysis
• Cell biology: microinjection into mammalian cells
,• Biotechnology: large-scale production of growth factors and hormones (eg.
interferon, insulin etc.)
• Pharmaceutical industry: develop screening assays for drug discovery programs
• Clinical: production of vaccines, diagnostic kits
1. Expression, Purification, Analysis of Recombinant Proteins
• For a long time, isolating protein was difficult – had to purify directly from a
tissue source, complexity of intact tissues and organs required long series of
chromatography steps – often yielded only few milligrams of pure protein
• For structural and functional analyses – lots of protein needed
• Nowadays – proteins can be grown in cell culture through genetic engineering
techniques
Protein expression techniques/systems
Expression in Prokaryotes
Bacterial (Vectors for constitutive and inducible expression)
Expression in Eukaryotes
Yeast (vectors: autonomously replicating and genome integrating)
Insect cells (baculoviruses)
Mammalian cells (transient transfections, stable cell lines, adeno-and retroviruses)
Aspects of bacterial expression Aspects of eukaryotic systems
- In bacteria many recombinant - Lower levels of expression (in most
proteins are misfolded, insoluble or cases)
degraded - More expensive (expression medium)
- Numerous post-translational - More complex and takes longer to
modifications do not occur in establish (~2 months for stable cell
bacteria, such as phosphorylation, line)
glycosylation
- Difficult to purify proteins >60kDa
Purification
• First step involves breaking open cells to release contents cell homogenate
- Break cells with high-frequency sound
- Mild detergent
- Force cells through small hole using high pressure
- Shear cells between close-fitting rotating plunger and thick walls of a glass vessel
• Centrifugation techniques to obtain proteins
, Protein separation techniques
a) By size
- Gel electrophoresis
- Gel filtration chromatography
- Ultracentrifugation
- Dialysis
b) By charge
- Isoelectric focusing
- Ion exchange chromatography
c) By polarity
- Paper and reverse-phase chromatography
- Hydrophobic interaction chromatography
d) By specificity
- Affinity chromatography – commonly used
Types of Electrophoresis
a) Filter paper
electrophoresis
- Proteins easily
denatured due to
high absorbance of
filter paper
- Works for small
peptides or amino
acids
b) Thin layer
electrophoresis
(TLC)
- Chemically-modified
cellulose
c) Gel electrophoresis
- Starch gel
electrophoresis
- Agarose gel electrophoresis
- Polyacrylamide gel electrophoresis (PAGE)’
Native-PAGE SDS-PAGE
• Enzyme activities are retained after • SDS (sodium dodecyl sulphate) coats
