CH4303 Bioseparations

Jiang Rongrong Lecture 1

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Syllabus

Objective: Learn downstream separation and purification of biological products, especially proteins and enzymes. The following topics are going to be discussed: sedimentation, centrifugation, membrane separations, cell disruption, electrophoresis, precipitation, leaching. Instructor: Dr. Jiang Rongrong (first half) N1.2-B1-08, rrjiang@ntu.edu.sg, 6514-1055

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Textbooks

1. “Biochemical Engineering” Harvey W. Blanch and Douglas S. Clark, Marcel Dekker, New York, 1997; ISBN: 0-8247-0099-6 (HD) (Chp. 6)

2. “Biocatalysis” Andreas S. Bommarius, Bettina R. Riebel, Wiley-VCH, 1st Edition, ISBN 3-527-30344-8, E-book available on NTU website (Chp. 8)

3. “Bioseparations Engineering” Michael R. Ladisch, Wiley-Interscience 2001, ISBN 0-471-24476-7

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Syllabus

Week 1 (Jan.15) Introduction & Recombinant DNA technology

Week 2 (Jan. 22) Recombinant DNA technology Week 3 (Jan. 29) Recombinant DNA technology Week 4 (Feb. 5) Sedimentation & Centrifugation Week 5 (Feb. 12) Cell Disruption Week 6 (Feb. 19) Off, British High Commission Week 7 (Feb. 26) Membrane Separation (I) Week 8 (Mar. 5) Off , recess

Syllabus

Week 9 (Mar. 12) Membrane separation (II) Week 10 (Mar. 19) Electrophoresis Week 11 (Mar. 26) Precipitation Week 12 (Apr. 2) Leaching Week 13 (Apr. 9) Review

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Outline

• Introduction on Bioseparations • Categories of purification methods • Important things in enzyme purification • Recombinant DNA Technology on protein

recovery

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Money, Money, Money

• Biotechnological process: – Medium preparation; Reaction; Downstream

processing • Separating and purifying product from the

fermentation broth or cell culture is very costly. – Normal ratio of costs: Fermentation: Product Recovery =60:40 – Recombinant DNA fermentation products: 80-90%

of the total manufacturing costs

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Steps of a biotechnological process

Biotechnological process:

1. Medium preparation

2. Fermentation

3. Downstream processing

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Reasons for High Costs

1. Product in small amounts To be separated from intact cells, soluble extracellular

products and residual substrates

2. Require several discrete steps, which depends on A) original materials B) concentration and physicochemical properties of product C) final purity required

Objective: Satisfy the purity requirements and minimize cost

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How to formulate a purification strategy?

Ans: consider A+B+C No.1--consider (C): What’s the required purity of the product?

– Allowable ranges of impurity concentrations – the specific impurities Example: if use recombinant DNA-derived

proteins in final therapeutic applications, <0.1% protein impurities; <100pg nucleic acid per dose (US standard, 1990)

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Purity Criteria

Once the purity Criteria selected, the specific purification procedures can be selected, and… there are other things to consider.

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Example: Recombinant proteins from living cells

• Recovery sequence depends partly on the characteristics of the host cells

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Categories of Purification Methods I

• Separation of insoluble: sedimentation, centrifugation and filtration – Insoluble (whole cells, cell debris, pellets of

aggregated protein, undissolved nutrients) • Isolation and Concentration: extraction,

ultrafiltration, precipitation and ion-exchange – Objective: Isolation of the desired product

from unrelated impurities

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Categories of Purification Methods II

• Primary Purification: chromatography, electrophoresis – More selective than isolation (e.g. distinguish

species with similar chemical and physical properties)

• Final Purification: crystallization, drying – When extremely high purity is required for

pharmaceuticals and therapeutics

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Example: Purification of recombinant interferon and antibiotics

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Important things to be managed during enzyme purification I

• Purity: comparing specific activity (U/mg enzyme) at the processing stage in question to the specific activity of the pure enzyme – Purification factor: specific activity at the

processing stage in question to the initial specific activity in the crude extract

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Important things to be managed during enzyme purification II

• Yield: a percentage of the amount of protein at the processing step in question compared to the amount of target protein contained in the crude extract after homogenization.

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Example: purification of an enzyme

1kDa

10kDa

15kDa

30kDa

50kDa

70kDa

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Purification Factor and Yield

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General Aspects of Bioproduct Recovery I

• Diverse products of biotechnology: cells OR specific metabolites of the cell – Two categories of metabolic products: Intracellular

OR extracellular (major impact on the purification scheme)

– Intracellular: vitamins, certain enzymes, a few antibiotics (e. g. sisomicin, griseofulvin)

– Extracellular: citric acid, alcohols, hydrolytic enzymes (e. g. proteases), most antibiotics (e. g. penicillin, streptomycin)

– Purification methods for intracellular and extracellular products are different

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General Aspects of Bioproduct Recovery II

• Low concentration bioproducts coupled with large amounts of interfering species can seriously complicate the task of purification

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Recombinant DNA Technology (Protein Recovery)

• Recombinant DNA technology has enabled the production of many important proteins that were previously extremely difficult or impossible in commercial quantities

• Example: 400g of growth hormone (for 550 patients) generated from 4000 L of E. coli fermentation in 1989; previously from 100,000 cadaver (corpse) pituitary glands

• Why are we interested in recombinant DNA technology: – higher production levels of the desired protein compared to the

natural source – create opportunities for genetic manipulations to facilitate

recovery

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World-wide manufacturing demands for recombinant DNA derived proteins

1. Gram to kg scale: Tissue Plasminogen Activator (tPA); the colony stimulating factors

2. Kilogram scale: therapeutic proteins as insulin (Genentech), human growth hormone, erythropoeitin, monoclonal antibodies

3. Multi-ton scale: for agriculture use such as porcine, bovine and ovine growth hormones

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IB on SDS-PAGE Gel

Soluble Insoluble inclusion body

Figure: Soluble and insoluble proteins after gene over-expression

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Recombinant DNA technique: Fusion Proteins

• Fusion Protein: --linking a small piece of gene with a

bacterial gene to produce protein – Why need it? Ans: Easy for purification -- How to do it? Ans: construct fusion proteins on the gene level

with tags

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Important things for designing fusion proteins for purification

• 1. Suitable for simple, rapid and inexpensive ion-exchange/affinity chromatography methods

• 2. efficiency of the linker peptide cleavage must be evaluated

• 3. a negligible effect on the target protein’s folding & no effect on activity

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Example: Fusion Protein 6xHis tag for Purification

How fusion protein works?

6xHis is encoded in the gene

Purification method: Immobilized Metal Affinity Chromatography

DNA Recombinant Technology

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Isolation of a specific DNA fragment of interest (e.g. a gene), and propagating identical copies (clones) of it in a suitable vector. This is achieved using recombinant DNA technology. **Common lab techniques (cloning, polymerase chain reaction, sequencing) are only possible because we learned how bacteria replicate and repair their DNA

DNA Recombinant Technology (Gene Cloning)

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BamH1 BamH1

BamH1

Bam

H1

Transform E. coli

BamH1

INSERT

VECTOR

Gene Cloning

Screening

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Restriction endonucleases in cloning

Restriction endonucleases recognize and cleave specific DNA sequences

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Where do restriction endonucleases come from?

Usually isolated from microbes Named after the microbes they are isolated from

EcoRI – isolated from E. coli strain R HindIII – isolated from Haemophilus influenzae strain Rd

Nobel Prize in 1978 was awarded for discovery of restriction enzymes

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If microbes produce these enzymes, why isn’t the DNA

digested by them? Restriction endonucleases protect microbes from invasion by foreign DNA

Microbes use methylation to protect their chromosomal DNA; invading viral DNA is not methylated Many restriction enzymes cannot cleave methylated DNA

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Examples Of Restriction Enzymes

35 New England Biolabs: http://www.neb.com

Theoretical frequency of restriction sites within a sequence depends on the number

of bases in the recognition sequence.

4 bases: 5: 1/1024 6: 1/4096 8: 1/65,536

Probability of finding a target of:

1/256

36 Restriction Enzyme website: Webcutter2.0 http://rna.lundberg.gu.se/cutter2

Restriction enzymes usually function as homodimers

G A A T T C

C T T A A G

5'

5' EcoRI: a cartoon view 37

Why are restriction enzymes so useful?

They cleave at a specific sequence, and often leave a single stranded overhang (“sticky end”)

The overhang can be either 5’ or 3’

Sometimes restriction enzymes leave a “blunt” end

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Sticky ends

5’-TCAGATCGTACTTGAG 3’-AGTCTAGCATGAACTCTTAA

AATTCGGGCT-3’ GCCCGA-5’

EcoRI

5’-TCAGATCGTACTTGAGAATTCGGGCT-3’ 3’- AGTCTAGCATGAACTCTTAAGCCCGA-5’

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5’CTAGGACCGAATTCAAGTACGGACC 3’ 3’ GATCCTGGCAATTGTTCATGCCTGG 5’

Fragment 2

5’ TCAGATCGTACTTGAGAATTCGGGCT-3’ 3’ AGTCTAGCATGAACTCTTAAGCCCGA-5’ Fragment 1

5’ TCAGATCGTACTTGAG AATTCGGGCT-3’ 3’ AGTCTAGCATGAACTCTTAA GCCCGA-5’

5’CTAGGACCG AATTCAAGTACGGACC 3’ 3’GATCCTGGCAATT GTTCATGCCTGG 5’

EcoRI

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5’TCAGATCGTACTTGAGAATTCAAGTACGGACC 3’ 3’ AGTCTAGCATGAACTCTTAAGTTCATGCCTGG 5’

A new recombinant DNA molecule

DNA ligase seals the nicks between the two strands, reforming covalent

phosphodiester bonds

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Restriction Enzyme

DNA Ligase

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Types of vectors 1. plasmids (small circular bacterial replicons) 2. viruses (bacteriophage, linear or circular) 3. linear artificial chromosomes (yeast only) Desirable characteristics of vector 1. stably replicates in host - needs origin of replication (ori) 2. easily introduced into host - usually into bacteria, sometimes yeast - by transformation (plasmid) or infection (virus) 3. marker to select cells containing vector - usually antibiotic resistance gene 4. good sites for inserting "cloned" DNA

Cloning Vectors

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A modern plasmid cloning vector (E. coli)

E. coli origin of replication

Ampicillin resistance gene

Multiple cloning site (polylinker) 44

BamH1 BamH1

BamH1

Bam

H1

BamH1

INSERT

VECTOR

Step 1: Ligation

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Ligation check: restriction mapping

BamH1 BamH1

BamH1

Bam

H1

BamH1

At least 4 possible products of ligation; These can be distinguished by restriction mapping (once cloned into E. coli)

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EcoRI restriction digests of DNA

from clones (#1-5) containing

inserts vector

Insert

Vector EcoRI EcoRI

Identifying the “correct” insert

3 kb

6 kb

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Why can we visualize the DNA on the gel?

Ethidium (fluorescent dye)

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BamH1 BamH1

BamH1

Bam

H1

Transform E. coli

BamH1

INSERT

VECTOR

Step 2: Transformation

Screening

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Bacterial transformation 1. Definition: acquisition of new genetic markers by incorporation of added DNA. 2. Two common methods for E. coli: chemical method and electroporation.

Circular DNA plasmid + calcium

-

-

- -

- Ca2+

"competent" bacterial cell

Ca2+ Ca2+

Transformed cell

-

-

- -

-

DNA Open "pores" in electric field: electroporation 50

BamH1 BamH1

BamH1

Bam

H1

Transform E. coli

BamH1

INSERT

VECTOR

Step 3: Screening

Screening

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Screening against vector lacking inserts

"blue-white screen" using ß-galactosidase (lacZ) gene

origin of replication

polylinker

LacZ gene

Ampicillin resistance gene

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Blue= no insert (lacZ+), white= insert (lacZ-, due to disruption of gene in plasmid)

ß-galactosidase gives blue reaction product:

Substrate X-gal: modified galactose sugar

ß-galactosidase can digest X-gal and form an insoluble blue product

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Clones: Copies

Gene

Grow Colony

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Nobel prize, 1993

POLYMERASE CHAIN REACTION (PCR)

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In polymerase chain reaction (PCR), multiple copies of DNA are synthesized using: - Template DNA - Oligonucleotide primers (designed with restriction sites) - DNA polymerase (e.g. Taq, pfu) - dNTP (dATP, dGTP, dCTP, dTTP)

target

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(longer than target region)

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Denature (94°C)

Hybridize primers (35-65°C)

(based on Tm)

DNA synthesis (72°C) (DNA pol from

Thermus aquaticus)

PCR, con't

Ca. 35 cycles 58

Geometric progression: after 40 cycles, theoretical product amplification of 240 = 1012 times (visible on gel)

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Some Applications of PCR

1. Rapid laboratory tool 2. DNA fingerprinting Forensic applications (crime scenes)

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