#BIOLOGY SERVICES BIOLOGY SERVICES : PROTEIN & MOLECULAR
“PROTEIN SCIENCE
Recombinant protein expression in different expression systems
1. Gene engineering
1.1 Overlap extension technique
1.2 Site-directed mutagenesis technique
1.3 Other PCR techniques
2. Protein production & characterization
2.1 purification and refolding of soluble, membranous and multimeric proteins
3. Testing
3.1 Affinity and functional assays
3.2 In cellulo studies
3.2 In vivo studies
3.2 Signalization studies
“METHODOLOGY
✔️ Circular dichroism
✔️ Fluorescence technique
✔️ Electron microscope
✔️ Subcellular localization based on cellular fractionation
✔️ Total protein dosage
✔️ In vitro propagation assays
“PROTEIN SCIENCE
Recombinant protein expression in different expression systems
1. Genetic engineering
1.2 Construction of one or more genes by PCR
Generation of homogenous or heterogenous constructs by overlap extension PCR.
1.3 Generation of protein mutants by PCR site-directed mutagenesis
1.4 Insertion of constructs into the expression plasmids
2. Bacterial expression
2.1 Transformation into an appropriate bacterial host and preparation of glycerol stocks
2.2 Optimization of protein expression by fine-tuning of bacterial growth rate and temperature
2.3 Characterization of protein expression in the soluble form or inclusion bodies
3. Protein purification
3.1 Native purification of the soluble proteins
3.2 Refolding purification from inclusion bodies
“MOLLECULAR
6. Cloning techniques
7. Different types of PCR
7.1 Reporter gene assays
7.2 Genomic and plasmid DNA isolation
7.3 Residual mammalian DNA detection by hybridization analysis
7.3 Total RNA isolation
8. Assay feasibility, development, and validation
9. Biological sample analysis
“METHODOLOGY
✔️ Circular dichroism
✔️ Fluorescence technique
✔️ Electron microscope
✔️ Subcellular localization based on cellular fractionation
✔️ Total protein dosage
✔️ In vitro propagation assays
