Production of Recombinant Proteins

● Protein Production in Prokaryotic Host

● Regulation of transcription
● Increasing translation efficiency
● Increasing protein stability
● Increasing protein secretion
● Facilitating protein purification
● DNA integration into the host chromosome

Production of Recombinant Proteins

● Protein Production in Eukaryotic Hosts

● Posttranslational modification of eukaryotic​ ​proteins
Eukaryotic expression systems (general features, ​yeast, baculovirus-insect
cells, mammalian cells)
● Protein Engineering
● Directed mutagenesis
● Random mutagenesis
● Examples of protein engineering

Increasing Protein Stability

1. By facilitating protein folding (e.g., low temperature or ​coexpress with genes

encoding proteins that function in​ ​proper protein folding such as chaperones)

2. By decreasing protein degradation (e.g., add specific amino ​acids to the

N-terminus to reduce protease digestion or ​express the protein as a fusion
protein)

Protein Production in Eukaryotic Hosts

• Unlike prokaryotes, eukaryotes carry out a number of posttranslational
modifications (PTMs) of proteins, which are often critical for proper protein
function

• These PTMs include:

• Glycosylation

• Processing of propeptides

• Phosphorylation

• Acetylation

• Methylation

• Hydroxylation

• Sulfation

• Acylation

• g-Carboxylation

• Myristoylation (C

• Palmitoylation (C1416

• Disulfide bond formation

fatty acid addition) fatty acid addition)