l. 5: Engineering Beyond Genetics

Problems in the protein production pathway?

proteolysis: secreted proteases in fungi and yeast can cut the product in the medium

How to solve proteolysis?

Which levers are there in eukaryotes?

promoter, signal peptide, carrier fusion,

What stresses the cell?

• the product (toxic / aggregating), high titres

• inhibitors in the medium

  • component released when biomass pre-treated (furfural, acetic acid from hydrosylate)

  • solvents

  • by-products (acetate)

• process conditions: pH, temperature, osmotic and oxidative stress

How to engineer tolerance?

What does N-glycosylation help with + example?

• folding, solubility, half-life, recognition

• therapeutic antibodies, erythropoietin

What does O-glycosylation do? + example

• protection, binding, solubility

• mucins, many secreted proteins

HEK

human embryonic kidney cell line

How to account for glycosylation?

• choose a human-like host (CHO / HEK)

• glycoengineered yeast

• remove a site (N → Q) to avoid glycosylation

• add glycosylation sites (prolongs half-life)

• 
  

• enzymatic remodelling of glycans after purification

s. 23 A

disulfide bond is the key reason, yeast / mammalian host cell

s. 23 B

e. coli is cheap, bacillus / fungi / yeast so it can be secreted, remove proteases

s. 23 C

mammalian cell,

s. 23 D

e coli, yeast, bacillus

inteins

protein-level introns

removes itself and joins the flanking parts

spytag / spycatcher

• most widely used

sortase

click chemistry

How to check

secretome

the complete collection of bioactive molecules (proteins, growth factors, cytokines, extracellular matrics components, membrane-bound extracellular vesicles) cells secrete into the extracellular space