Steps of Protein Production

What is the first step of protein production?

determine the gene (nucleic acid) sequence (NCBI) that encodes desired protein

What is the second step of protein production?

obtain the best source for nucleic acid (message)

What are the best sources for nucleic acid (3)?

(1) gene synthesis, (2) genomic DNA, (3) mRNA and cDNA

What is the third step of protein production?

clone the gene into a vector

What is the fourth step of protein production?

Introduce the clone vector into the proper living cell and fermentation

What are the different living systems for producing biopharmaceuticals (3)?

prokaryotes (E. coli), Yeast, Mammalian cells (CHO, HEK293, insect)

What are the pros to prokaryotes (E.coli) (7)?

easy manipulation, rapid growth, large scale fermentation, simple and low cost media, high yield, low cost, suitable for small proteins

What are cons to prokaryotes (E.coli) (3)?

almost no post translational modifications (PTM), may aggregate or not fold properly, not appropriate for large proteins

Where is protein production done in prokaryotes (E.coli)?

inside cell

What are the pros with using yeast (3)?

relatively rapid growth, large scale fermentation, performs some PTMs

What are the cons with using yeast (1)?

does not perform all PTMs or performs them differently

Where are the produced proteins found in yeast?

sometimes in cells, sometimes in media (?) (just says sometimes in chart)

What are the pros with using mammalian cells (2)?

appropriate for large proteins, performs all PTMs

What are the cons with using mammalian cells (5)?

slow growth, expensive media, difficult to perform large scale, requires more technical expertise, high cost (> $1 million/kg)

Where are the produced proteins found after production via mammalian cells?

secreted in media