Suppose you have the sequence for a gene, how will you find the protein?
Use in silico methods on the ORF - BLAST
Then design a primer based on the ORF and clone it.
* Overall starting from a gene sequence and trying to find its expression
* Called reverse genetics
* Start from a sequence, find the ORF, go through protein expression steps, and find out the gene expression.
* Forward genetics is going from the phenotype to the sequence.
In what host do you express a recombinant protein expression?
you express it in a bacteria
* E.coli is the best
* Yeast can be used
Define Heterologous expression
when the DNA and host are from two different sources.
What are 2 sources of problems of expression?
From the gene (ORF) and host, when two they are from different sources.
Transcription signal - the genes have a different transcription signals (nucleotide sequences)
Codon bias - E.coli has a preference to use a specific codon to code for a specific amino acid.
What issues can happen if the gene (ORF) and host are from different sources. How is it avoided?
intron issues - the bacteria cannot remove the introns
* Prokaryotes do not have the mechanisms to splice the introns
* Ecoli cannot splice introns, does not have splicesomes. The mRNA will not be able to be translated.
* To avoid this issue, use cDNA, a complementary DNA to the mRNA.
What happens if in expression, the transcription signal is an issue?
- The gene sequence could be signaled as a transcription stop site
- Results in an immature transcript, small transcripts generating no protein or the incorrect protein.
What are 3 host issues in expression?
- No secondary modification
- No disulfide bond formation
- Presence of inclusion bodies
What happens if there is no secondary modification?
- In secondary modifications/ posttranslational modifications (glycosylation, phosphorylation)
- Due to no structural/ conformational change, the protein will not have the proper structure resulting in the protein being non-functional.
What happens if there is no disulfide bond formation?
- The cytosol in the bacterial cells is a reductive environment, meaning no bonds are made.
- Bonds are made in a oxidative environment
What is the presence of inclusion bodies?
- Insoluble aggregates of proteins
- Always a problem in recombinant expression
- May stop expressing protein for recombinant expression
- Proteins will aggregate to make more space for overpressed protein.
What are the 3 Advantages of inclusion bodies?
- Easy to purify: centrifuge the culture after lysis due to size of protein and they are relatively pure.
- Protected from proteolytic cleavage: The enzyme proteases (enzymes degrading protein). The proteases are relatively inactive but become active when purification occurs. Cannot be done in inclusion bodies because the cleavage sites are not exposed.
What are the 3 disadvantages of inclusion bodies?
- Solubilization - difficult to make a soluble protein
- Can use a different type of buffer to make the pH one where the amino acids in the protein are soluble.
- Folding: may not be able to be refolded back to the original active protein. Makes the protein unusable.
- To rectify, need the pH, ionic strength (amount of salt in the protein), and good temperature.
- If these disadvantages are rectified, the inclusion body can have a function.
Should you expect inclusion bodies? What can be done?
In general, expect the form of insoluble bodies when overexpression occurs.
Inclusion bodies are a consequence of over expression to help with storing overexpressed protein.
Need to find another host
What type of expression is the Recombinant DNA?
Heterologous expression (two different DNA)
What are two problems caused by foreign genes? Give an example. What is the solution and how is it done?
Presence of introns
Ex: You are expressing beta-globin gene from human in E.coli
Problem:The beta globin gene has introns
Solution: Use the c-DNA
* use reverse transcription to get the c-DNA. Use reverse transcriptase.
What is codon bias? What is the solution?
- Prefer codons correlate with abundance of related tRNA available in the cell
- If there is an issue of codon bias, there is no availability of related tRNA resulting in no translation and no protein.
- There are certain codons that will code certain things in your foreign gene that will not correlate to codons in the host. Therefore there will not be any translation since those codons cannot be translated by the host.
- Solution: overexpression of tRNA for the codon with the bias.
