Industrial Biotechnology

Lecture 9. Protein expression and production

 Expression systems
• Bacterial
• Fungal/yeast
• Animal
• Plant
• Insect
• Other

 rProtein recovery
• His tags
• MBP fusions
 Objectives of protein expression in
industrial production

 Safe and controllable fermentation

 Cheap fermentation
 High level of recombinant production (g/L not
mg/L)
 Easy recovery of expressed proteins
 No degradation of expressed protein
 Microbial expression hosts – E. coli

ADVANTAGES DISADVANTAGES
 Numerous specific vectors  Not naturally competent
( phage and pUC plasmid  Low expression yields
derivatives)
 No glycosylation of rProtein
 Very high transformation
 rProtein not exported
efficiencies
 Very well understood
fermentations
 Simple cell recovery and lysis
A typical E. coli plasmid vector

• Blue/white screening
• SP6 or T7 RNA polymerase promoters
• Amp resistance selection
 Microbial expression hosts –
Saccharomyces cerevisiae
ADVANTAGES DISADVANTAGES
 Moderate expression yields  Low-medium transformation
 rProtein glycosylated efficiency
 rProtein exported  Limited range of vector systems
 Very well understood  rGenes must be stably
fermentations incorporated
 Simple cell recovery and lysis
Microbial expression hosts – Pichia pastoris
and Kluveromyces sp.
ADVANTAGES DISADVANTAGES
 Moderate expression  Low-medium
yields transformation efficiencies
 rProtein glycosylated  Difficult fermentations
 rProtein exported  Limited range of vector
 Simple cell recovery systems
 rGenes must be stably
incorporated
 Unusual codon usages
Microbial expression hosts – Trichoderma
reesii
ADVANTAGES DISADVANTAGES
 High expression yields  Low-medium
 rProtein glycosylated transformation efficiencies
 rProtein exported  Difficult fermentations
 Simple cell recovery  Limited range of vector
systems
 rGenes must be stably
incorporated
 Unusual codon usages
 Microbial expression hosts – Bacillus

ADVANTAGES
 Very high expression yields
(>10 g/L)
 Simple fermentations
 rProtein exported
 Simple cell recovery
 Naturally competent
DISADVANTAGES
 Limited range of vector systems
 Optimised vector systems are
proprietary
 No glycosylation
 Variable transformation
efficiencies
 Insect cell expression
(Specific host-vector system; Sf9 cultured insect cells and
Baculovirus vectors)
 Advantages  Disadvantages
 Higher eukaryote-like  Highly specific
glycosylation transformation system
 Scaleable to large volumes (Baculovirus)
 Fastidious culture
 Very slow doubling time
(20h)
 Animal cell culture
 Advantages
 Exact glycosylation
 Can be transformed to
continuous cell lines
 Some (e.g., CHO cells)
grow in suspension culture
 Some carcinoma-derived
cells (e.g., HeLa cells) have
rapid growth rates
 Extracellular expression
 Disadvantages
 Some grow as adherent
layer (anchorage dependent)
 Fastidious growth
requirements
 Very slow doubling time
(15-25h)
 Very susceptible to
contamination
 Very sensitive to shear
stress
 Plant cell culture
 Advantages
 Suspension cultures
 Low aeration required
 rProtein directed to vacuole
 Disadvantages
 Restricted range of
transformation systems
• Agrobacterium for dicots
• Biolistics for monocots
• Specific plant viruses
 Very slow doubling times
(15h – 48h)
 Very sensitive to shear stress
 Grow heterotrophically but
require light for optimum
growth
 Other expression systems

 Tobacco plant expression

 Using recombinant tobacco mosaic virus
 Tissue specific promoters
 Scale-up of production as for normal agriculture

 Whole animal expression

 Transformation of unfertilized embryo
 Use of tissue-specific promotors (e.g., lactose-specific
promoter for expression of proteins in milk)
 Expression development

 Multicopy plasmids
 Multiple selective (resistance) markers (Ampicillin,
Tetracycline, Kanamycin, Thiostreptin)
 Different promoters (ITPG, Trp, temperature-inducible,
aTet)
 Multiple promoters (shuttle vectors for different hosts)
 Multiple ori sequences
 N-terminal tags (e.g., polyHis) with cleavage site
 Vectors designed for protein recovery:
His Tags
 Vector contains polyGCA
 rProtein has polyHis Tag
 Recovery with Ni-NTA
affinity matrices
 Vectors designed for protein recovery:
Maltose Binding Protein

 Cloning in pMAL vector generates fusion protein with

MBP, with protease target site in fusion linkage
 Cytoplasmic expression
 Protein recovered using amlyose affinity column
 Fusion protein cleaved using Factor X protease
 rProtein recovered by affinity chromatography