Download - Lecture 5
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Lecture 5: Biotechnology
and prokaryotes
•Bacteria and bacterial systems;•Manipulation of gene expression in prokaryotes (Chapt 6); •Large-scale purification of proteins from recombinant microorganisms (Chapt 16)
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Biotechnology and prokaryotes
•“germs”•Prokaryotes are ubiquitous•Wide spectrum of species, types, characteristics•“Relatively simple” systems, for manipulation•Biology characterized•Genetics characterized•[Molecular biology characterized]•Genomics, bioinformatics, systems•Industrialization, [weapon-ization]•Two-edged blade: “anti- and pro-”•Effects on humans, other organisms, environment
•[medical (pathogen), therapeutic (food)]…•Less legal, ethical, moral issues
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Phylogeny and range of
prokaryotes
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Phylogenic branches
•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf
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Shapes and “multicellular”-ism
•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf
•quorum•fluorescence•biofilms
•‘differentiation’- Myxococcus xanthus
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•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf
Optimum growth temperature
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Structure of a type of bacterium
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Types of bacteria: gram positive vs
gram negative
Characterization of bacteria: Gram stain•Hans Christian Gram, 1882•Crystal violet -> methylene blue
•wikipedia
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Types of bacteria: gram positive vs gram negative
•Gram positive:•Thick cell wall, peptidoglycan•Examples: Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, Clostridium
•Gram negative:•Cell wall, lipopolysaccharide (also, LPS or endotoxin layer) plus [less] peptidoglycan•Examples: Escherichia coli, Salmonella, Pseudomonas, Legionella
wikipedia
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Bacillus types and biotechnology
Gram positive:•Contrast B. anthracis vs. B. thuringiensis vs B. subtilis•Spores vs crystals•Ag biotech
•http://www.scq.ubc.ca/wp-content/uploads/2006/08/GM-crop.gif•http://images.google.com/imgres?imgurl=http://www.magma.ca/~scimat/B_thur16.jpg&imgrefurl=http://www.magma.ca/~scimat/B_thurin.htm&h=324&w=340&sz=23&tbnid=4A1w35t4YFto8M:&tbnh=113&tbnw=119&prev=/images%3Fq%3Dbacillus%2Bthuringiensis%26um%3D1&start=1&sa=X&oi=images&ct=image&cd=1•wikipedia
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Bacillus and biotechnology
Gram positive:•B. thuringiensis•Spores vs crystals•Ag biotech
•http://www.scq.ubc.ca/wp-content/uploads/2006/08/GM-crop.gif•http://images.google.com/imgres?imgurl=http://www.magma.ca/~scimat/B_thur16.jpg&imgrefurl=http://www.magma.ca/~scimat/B_thurin.htm&h=324&w=340&sz=23&tbnid=4A1w35t4YFto8M:&tbnh=113&tbnw=119&prev=/images%3Fq%3Dbacillus%2Bthuringiensis%26um%3D1&start=1&sa=X&oi=images&ct=image&cd=1•wikipedia
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Wall-less bacteria
•Wall-less bacteria•Smallest, simplest free-living self-replicating organisms; still, complex•Reduced genome species•~100 species of bacteria and archaea•Membrane ~30% lipid and 70% protein•25-30% cholesterol, comparable to eukaryotes•[other bacteria do not have cholesterol or sterols in PM]•Example: Mycoplasma pneumoniae, Thermoplasma acidophilum
•L-form or cell wall deficient bacteria•Chronic disease? [rheumatoid arthritis, Chronic Fatigue Syndrome, Lyme disease]
•[http://bacteriality.com/2007/08/18/history/]
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Life on the Edge
•Deinococcus radiodurans (eubacteria)•Survives 5 Mrads
•DNA highly damaged at 1.7 Mrads; 1,000 to 2,000 DNA fgmts repaired in 24 hrs•RE map; Four genome elements, circular <-> linear large
http://www.ornl.gov/sci/techresources/Human_Genome/publicat/microbial/image3.html
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Life on the Edge
•Science Daily (Source: NASA, +), 4/4/00 “Weird Life”•The limits of life on Earth are much broader than previously thought. •Examples of life at extreme conditions include:
•Highest radiation: 5 million rads-- Deinococcus radiodurans (eubacteria)•Hottest: 235.4 F (113 C)-- bacteria from deep sea vents•Coldest: 5 F (-15 C)-- microalgae in Antarctic rocks•Deepest: Two miles underground in rocks-- bacteria•Most acidic: pH 0-- Unclassified organisms growing on gypsum in caves•Basic: pH 9.0; 12% salt-- bacteria, Soap Lake, Southeast WA•Saltiest: 30 percent salt environment-- bacteria•Deepest and Highest pressure: 1200 atm-- at bottom of Marianas Trench (ocean)•[Farthest: Moon-- Streptococcus mitus (from human source) from Surveyor III camera after three years unprotected on lunar surface]
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Varied growth rates,
oxygen needs
•TThiel, UMissouri•www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf
•20 min: E. coli•24 hrs: M. tuberculosis
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Manipulating bacteria
•http://www.mansfield.ohio-state.edu/~sabedon/black06.htm
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Bacterial growth
•http://www-micro.msb.le.ac.uk/labwork/bact/bact1.htm
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Bacterial growth: quantify
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Natural selection and
growth
•Thomas Malthus. 1798, “Essay on the Principle of Population”•“population would outstrip food supply”
•Charles Darwin. 1859, “The Origin of Species” •“an application of doctrines of Malthus”
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Natural selection and
growth: How?
•Glucose- depleted•Fructose- not available•Maltose- not available•Lactose- available
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Microbial genetics and
molecular biology
•Genes and pathways are under strict regulatory control
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Microbial genetics and molecular biotechnology
•Control timing of expression of foreign gene product•Lac promoter
•Repressed by lac repressor•Derepressed by lactose and IPTG•lacUV5 -nuc change in -10 region•Stronger promoter
•Trp promoter•Repressed by trp repressor•Derepressed by trp and indoleacrylic acid•“leaky”
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hybrid promoter: tac
•Overexpression of foreign gene product•eg, lac repressor protein:•~10 molecules per cell•‘need’ ~1 umol for biochem studies•1 umol = 6 x1017 molecules•Or 6 x1016 cells•E. coli at stationary phase has•~4 x108 cells/mL•*if* 100% recovery, need 150,000 L
•tac promoter•Hybrid of trp and lac promoters•Matches consensus for E. coli RNAP seq•HAdeBoer, et al. 83. PNAS 80:21
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hybrid promoter: tac[not always straight-
forward]
•tac promoter•tacI, tacII
•lacUV5 -nuc change in -10 region •Relative strengths
•tac/lac 11.8x; tac/trp 3.5x•Strain dependent•HAdeBoer, et al. 83. PNAS 80:21
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Bacteriophage genetics
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Bacteriophage genetics and molecular biology
•Lambda promoter•Temperature sensitive promoter•Repressed unless temperature raised•Overexpression of foreign gene product
•incompatible or lethal/deleterous
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dual promoters: lac and T7
•Repressed unless inducer is present•Overexpression of foreign gene product
•incompatible or lethal/deleterous
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Plasmids
•JLederberg, 1952. Physiol. Rev. 32: 403 -”plasmid”•Circular, extrachromosomal double-stranded DNA•Size: 1 kb to 400 kb•Number of copies: 1-2 to 20s to 100s ~ori
•wikipedia
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Plasmids
•Conjugation- horizontal gene transfer•vs ‘episome’ (plasmid that can integrate into chromosomal DNA)
•Size: 1 kb to 400 kb•Number: 1-2 to 20s to 100s ~ori wikipedia
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Overexpression strategy: construction of hybrid vector
•Have: pPLc2833•Strong promoter pL, selectable marker, MCS
•Have: pKN402 ori gives increased copy number 5-10x•Temperature-dependent copy number
•Construct: hybrid pCP3•pL promoter and ApR gene with high-copy ori
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Overexpression strategy: plasmid number increase
•Have: pPLc2833•Strong promoter pL, selectable marker, MCS
•Have: pKN402 ori gives increased copy number 5-10x•Temperature-dependent copy number
•Construct: hybrid pCP3•pL promoter and ApR gene with high-copy ori
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Large-scale prep considerations
•IPTG and other derepressing chemicals can become expensive•Temperature-dependent shifts: time and energy for large cultures•Reconfigure pL with trp promoter•Grow in molasses and casein hydrolysate, low in free trp; derepress with tryptone [crude extract]•ex, -galactosidase and citrate synthase genes-> overproduced to 21% and 24% of cellular proteins
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Other bacterial hosts
•Genetics and molecular biology not as well-developed as E. coli•Using E. coli system in other gram negatives•(Nm is neomycin resistance; S1 is R. meliloti ribo protein gene)
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Universal gram negative
vector
•70 bp fgmt from Tn-5 Terminal IR•Low copy-number broad-host-range plasmid•E. coli, Alcaligenes sp, Enterobacter cloacae, Klebsiella pneumoniae,•Pseudomonas stutzeri, Pseudomonas fluorescens, Serratia marcescens
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Modifications for human
consumption
•Lactic acid metabolizing bacteria, eg Lactococcus spp•Dairy products- cheese and yogurt•Desirable to increase yields, add to quality of food•Undesirable to alter production process, product palatability, appearance, etc.•Cannot add chemical inducers or temperature effects to processing•Consensus constitutive promoter--> synthetic constitutive promoters•Tested 36 constructs•Most active ~7,000x stronger than least active•If -10 or -35 regions intact, ~400x in strength
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Fusion proteins
•Not all heterologously expressed proteins are expressed•Not all heterologously expressed proteins are expressed ‘enough’•Not all heterologously expressed proteins are stable as expressed•Engineer a fusion protein that has a partial structure of a ‘host-native’•Increase expression•Extend half-life•Requires correct reading frame•Above, proteolytic cleavage by blood coagulation factor Xa
•Val is N-term of desired protein•Based on existing biology….
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Blood clotting
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Post-translational modifications
•Extend half-life•Address protein delivery•Stabilize structure•Enhance biological activity
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Vector for expression fused protein
•5’-terminal segment of ompF gene for producing antigen•Directs synthesis of outer membrane protein and part of -galactosidase•Omp provides signals for txn and tnl, and secretion of product•lacZ functional, as a reporter for in-frame•“tribrid” has all three products’ characteristics
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Purification protocol
enhancement
•Efficient protocols for purification of product
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Fusion construct example
•Product is a secretion fusion protein•Fusion is marker peptide plus interleukin-2•Marker protein serves to extend half-life and to enable rapid purification•One-step purification via immunoaffinity chromatography•Small marker peptide does not stress host resources•For FDA, removal specifically with bovine intestinal enterokinase, a protease
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Fusion protein purification
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Column chromatography/HPLC
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“Recombinant DNA era” protein purification
•DNFrick and MJBessman JBC 270: 1529 (95) •Over-expression and purification of the Orf257 protein: SDS-PAGE and PAGE
A1) markers; A2 and 3) -/+IPTG;A 4 and 5) 2 and 6 ug Fraction VB1) markers; B2 and 3) 2 and 6 ug Fraction V
•Protocol:1. DEAE-Sepharose2. Streptomycin-SO4 fractionation3. Sephadex G-100 chromatography
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Rapid screening methods:
bacteriophage
•Large libraries of recombinants- How to find clone of interest?•ex., cDNA library with 5 x1010 clones, proteins which are rarely occurring cDNAs
•Can fuse with a surface protein gene of either a filamentous phage or bacterium•After txn and tnl, fusion is expressed on the surface of the organism•Can screen with antibody
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Rapid screening methods: bacterium
•Large libraries of recombinants- How to find clone of interest?•ex., cDNA library with 5 x1010 clones, proteins which are rarely occurring cDNAs
•Can fuse with a surface protein gene of either a filamentous phage or bacterium•After txn and tnl, fusion is expressed on the surface of the organism•Can screen with antibody
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Using probes: practice, alternatives and updates(surface display)
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Overexpression of foreign product
•Increasing plasmid copy number sometimes reduces yield of product•Cell resources diverted to production of other plasmid-encoded products•Creation of tandem arrays of the product•In-frame expression of multiple copies•Each copy has own signals•Copy number limits yield as well; unstable inserts
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Translation expression vectors
•Requires more than regulated strong promoter•Efficiency of translation and stability of nascent peptide•Prokaryotes: proteins are synthesized at different efficiencies, up to 100x•One aspect- translational initiation signal or ribosome-binding site•If mRNA has hairpin, can disrupt•Above GGGGG is rbs and AUG is start
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Construction of a
translation efficient vector
•ApR marker•tac promoter•lacZ ribosome-binding site•ATG start site, 8 nucs downstream from rbs•Two txn terminators T1 and T2 from lambda
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Translation efficiency
•Different tRNA usage•AGG, AGA, AUA, CUA and CGA are least-used codons in E. coli•Engineer strains to have different pools•ex., h2 protein, a peanut allergen, overproduced 100x than in conventional strains
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Posttranslational
stabilization
•Half-life ranges from few minutes to hours•Posttranslational modification of amino acids, •Including addition of single amino acid to N-term•Certain strings of AAc internally can increase proteolytic degradation
•PEST, rich in pro, glu, ser, thr•[change these sequences?]
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Posttranslational problems: folding
•Insoluble proteins, esp when overexpressed•Non-biologically active•Fusion with 11.7 kDa thioredoxin allows target protein up to 40% of cellular proteins•Above, trp controls cI to shut target expression; no trp-> no cI -> target expression
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Peptide folding aid
•Overproduction of E. coli disulfide bond-forming protein (DsbC) on •Synthesis of active human tissue plasminogen activator•Normal levels DsbC vs overproduction of DsbC with overproduction of tPA•tPA addressed to periplasm
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Misc factors
•Oxygen limitation: aerobic, anaerobic, facultative anaerobic•Most microbes used for overproduction require oxygen for growth•Oxygen has limited solubility•Cell density can outstrip oxygen availability, leading to stationary phase•(oxygen is a resource, just as nutrients)•Esp in stationary phase, where degradative proteins are produced
•Protease-deficient strains•Develop strains that are deficient in degradative proteins•E. coli has >25 different proteases•Some are for house-keeping functions, eg removing abnormal and defective proteins•Most engineered strains are slow growers•One strain, rpoH- (heat shock synthesis sigma factor) and a gene for a protease-, secreted •Proteins with a 36x greater SA than from WT cells (indicates decrease in proteolysis)
•Bacterial hemoglobin•Some microbes, strains of Vitreoscilla -obligate aerobe-, live in oxygen-poor environments•For their oxygen needs, synthesize hemoglobin-like protein•Raises effective intracellular oxygen•Cloned into E. coli, more effective metabolism: higher levels protein synthesis, proton pumping,
higher ATP production rate, higher ATP concentration
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Strategy: integration into chromosome
•Plasmids impose metabolic loads•Energy for replication, txn, tnl•Leads to unstable inserts, plasmids; slow growing
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Chromosomal integration and expression
•B. subtilis•E. coli plasmid with B. amyloliquefaciens -amylase•Protocol to integrate into different predetermined sites on chromosome•At nonessential sites
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Genome integration
•B. subtilis•E. coli plasmid with B. amyloliquefaciens -amylase•Protocol to integrate into different predetermined sites on chromosome•At nonessential sites
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Removal selectable marker genes
•Selectable marker required for lab protocols•Release of antibiotics-resistance into environment not good•Cre is on a second plasmid in same cell; IPTG induced•Excision of marker
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Increasing secretion
•Addressing: signal peptide•Stability depends on location of product:•Proinsulin is 10x more stable if inserted into membrane
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Increasing secretion example: not simple
•Interleukin-2 fused with E. coli maltose-binding protein signal peptide•Requires a part of the MBP gene as well
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Other strategies for secretion
•L-form bacteria are variants that lack cell wall•Arise via spontaneous mutation or treatment
•Penicillin- inhibits final step in cell wall formation•Lysozyme- hydrolyzes cell wall saccharide bonds
•Can transform with engineered vector:•L-form P. mirabilis•E. coli alk p’ase leader peptide/ Leu-Gly linker/Pro7/T. vulgaris carboxypeptidase/ txn term signal
•Carboxypeptidase secreted maximally after 36 hrs fermentation
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Metabolic loads
•Overexpression of foreign protein
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Metabolic loads, example
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Lecture 5 (cont)
•Chapt 16•Large-scale production of proteins from recombinant microorganisms•Technical considerations
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Generalized scheme for lg-sc fermentation process
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Microbial growth
•Batch, fed-batch (staged addn of nutrients), continuous
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Bacterial growth
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Bioreactors
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Two-stage fermentation in
tandem
•Temperature-dependent induction of a protein product
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Fermentation vessels
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Fermentation vessels
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Fermentation vessels
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Two-stage fermentation, one vat
•Tripartite fusion protein AGgal•Encodes five IgG binding domains of S. aureus protein A plus two IgG binding domains from S. str G148 protein G plus B-galactosidase E. coli•Driven with Lambda promoter•5 L culture with Ap and Kn seeds 600 L sans both•In 600 L, temp shift 30 to 40
•Loses 50% plasmids after 4 hrs•After 4 hr, yield at ~20% dry wgt of total biomass
•Saves on space, antibiotics, time (did not have to integrate into chromosome)
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Batch vs fed-batch
fermentation
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Batch vs fed-batch
fermentation
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Harvesting cells: Bucket centrifuge
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Harvesting cells: Sharples centrifuge
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Harvesting: continuous
membrane filtration
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Cell disruption
•Wet milling•High-pressure homogenization•Impingement•[sonication]•[enzymatic]
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Downstream processing
•After disruption,•Cell debris removal by centrifugation or membrane microfiltration dialysis•One membrane, cut-off 10 kDa
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Downstream processing
•After disruption,•Cell debris removal by centrifugation or membrane microfiltration dialysis•Two membranes, differential filtration