Lecture 8 Flashcards
What are the sources of steroid hormones? Describe the structure of steroid hormones and their synthetic analogues
Natural sources = plants, microorganisms, bile salts
Artificial sources = synthetic
- bind to receptors = agonists/antagonists with high affinity
- more stable (longer half life, degraded more slowly)
- generally more potent than natural
Steroids more stable than proteins
3D structures make them acive
How is the sequence of short peptide hormones determined and how can they be synthesized?
- sequence peptides from NH2 end by Edman degradation
Determination of AA sequence from DNA sequence:
- if gene is known, read the open reading frame and deduce the AA sequence from the genetic code
- if gene is unknown but large protein is purified –> sequence first 10-20 AA, use corresponding DNA sequence to synthesize probe, screen a cDNA library (made of mRNA), sequence corresponding cDNA and deduce full AA sequence
Helical synthesis of peptides:
- AA = bifunctional - must block groups that are not involved in formation of peptide bond
- need to set direction (which NH2, which COOH) –> protects other group, forms peptide bond; use solid phase (1st AA attached to resin)
= high cost, limited length (max 30 AA, limited recovery)
What is the relative purity of steroid versus protein hormone preparations? What is the relative purity of hormone preparations from recombinant and natural sources?
a. Advantage of recombinant DNA technology methods for producing proteins → lower cost, high efficiency and the production of a higher-purity, consistent product
b. In vitro recombinant DNA undergoes purification = high purity
Describe mechanisms whereby synthetic hormones can be more potent than natural source hormones.
Bind to receptors with high affinity and are more stable (e.g. degraded more slowly, longer lasting) = more potent
Outline strategies to determine the amino acid sequence of proteins by recombinant DNA methods.
a. Build sequence ‘in silico’ from ESTs or isolate protein and determine partial sequence
b. Isolate mRNA from tissue that expresses protein
c. Reverse transcribe to form cDNA
d. PCR amplification of cDNA for protein using specific primers
e. Sequence cDNA and deduce AA sequence
What are peptidomimetics or peptoides and what are their advantages compared to peptide hormones?
a. Act as agonists or antagonists for peptide hormones
b. Mimic the 3D structure of the hormone - same shape but chemically different
c. Advantages
- Unlimited variety of compounds can be made with various biological activities
- Much more stable and can get across cell membranes
Outline strategies for the synthesis of protein hormones using recombinant DNA.
a. Isolate and sequence gene/mRNA
- Need to have previous knowledge of the hormone
b. Clone it in a expression factor
- Choice of apprpriate vector
- Construct need to stay in cell
c. Introduce construct in bacteria/cells
- Gene needs to be expresses and protein synthesized
- Product secreted? Intracellular?
d. Recover/purifiy product
- Consider using fusion protein or tag for affinity purification
- Bacteria do not modfy proteins after translation
e. Test activity and safety
- Bacteria always start protein with methionine (potentially immunogenic)
- What about signal peptide (pro-hormone)?
- Post translational modification
