1. Protein engineering and recombinant protein expression

2. Protein biotechnology or engineering
Definition:
Deliberate design and production of proteins with novel or altered structure and properties, that are not found in natural proteins.

3. Central Dogma of Molecular Biology
This week: Molecular Biology Module
Proposed by Francis Crick in 1958
Oversimplification b/c RNA can RT to DNA; RNA can replicate itself, noncoding RNAs, alternative splicing, introns, etc.
Molecular biology is the study of the process of replication, transcription and translation of the genetic material
Interesting fact: Crick did not have a PhD at the time of his discovery, switched from physics to mol biol after a WWII bomb destroyed his lab equipment.
Proposed by Francis Crick, 1958

4. Applications Functional Studies Enzymatic Assays
Protein-protein interactions
Protein structural Studies
Protein Crystallography & NMR Structure Determination
Target Proteins for Rational Drug Design
Therapeutic Proteins – Preclinical Studies

6. Principle in recombinant protein expression
Bioinformatics
Target identification and cloning
Protein purification and production
Protein expression test
Applications
Applications

7. Things need to be considered for recombinant protein expression:
How to produce?
choose for protein expression system (vector and host)
2. How to make an expression recombinant DNA construct?
translational or transcriptional fusion, promoter use (inducible or constitutive)
3. Where to express?
cytosol, periplasm, secretion, inclusion body
4. Difficulties (protein expression problems)

10. Which host cell expression system?
E. Coli
Yeast
Insect cells
Mammalian cells
Cell free

13. Choose of protein expression system
The KEY idea is the cloned gene must be transcribed and
translated most efficiently.
Expression vector: MAXIMIZE GENE EXPRESSION.
Host: MINIMIZE TURNOVER OF GENE PRODUCTS
(preventing proteolysis in vivo in E. coli).

15. Protein expression in E. coli
pGEX plasmid:
Gene encoding affinity tag-glutathione S tranferase (GST)
Spacer between genes - encodes protease cleavage site (thrombin)
Ptac promoter-induce with IPTG
Ribosome binding site

16. Architecture of reporter gene constructs
(A) Transcriptional reporter, (B) translational reporter
Transcriptional reporters consist of a promoter fragment from a gene of interest driving GFP (Figure 1A). Typically, promoter fragments of a few kilobases immediately upstream of the start codon contain a significant portion of the cis-regulatory information necessary to provide a tentative expression pattern of the endogenous gene under study.
Translational reporters are in-frame gene fusions between GFP and a gene of interest (Figure 1B). Ideally, a translational reporter includes the entire genomic locus of a gene (5’ upstream region, exons, introns, 3 UTR). GFP can be inserted at any point in the open reading frame, preferably at a site that does not disrupt protein function or topology.

17. Increase selectivity of protein purification: (Gene fusion strategies)
Most target protein lack a suitable
Affinity ligand usable for capture on
a solid matrix. A way to circumvent this
obstacle is to genetically fuse the gene
encoding the target protein with a gene
encoding a purification tag. When the
chimeric protein is expressed, the tag
allows for specific capture of the fusion
protein. This will allow the purification
of virtually any protein without any prior
knowledge of its biochemical properties.
Hearn and Acosta, 2001

18. Cell lysis Cell lysis: rupture cell wall / plasma membrane,
--> release contents (organelles, proteins…)
1. Physical means
2. Sonication
3. Osmotic shock

19. Differential Centrifugation

20. Protein purification – column chromatography
Protein mixture applied to column
Solvent (buffer) applied to top, flowed through column
Different proteins interact with matrix to different extents, flow at different rates
Proteins collected separately in different fractions

21. General problems with gene expression
(a) Not enough protein is produced:
- codon usage preferential (rare codon)
- potential mRNA secondary structure.(5’-end ATcontent,
3’-end transcriptional terminator)
- toxic gene.
(b) Enough protein is produced, but it is insoluble:
* vary the growth temperature.
* change fermentation medium.
* low-copy-number plasmas.
* selection of promoter.

22. OPTIMIZING TRANSCRIPTION OF THE CLONED GENE
1. genetic fusion to strong promoters (transcriptional fusion).
increased gene dosage (utilize the gene’s
own promoter with the gene on a high-copy plasmid).
3. potential problem with toxic genes and available methods for efficient repression.
4. solutions to potential problems with premature termination and mRNA instability.