Week 5: Gene Cloning and Expression in Bacteria

Examples of therapeutic, proteins

• hormones

• blood factors

• insulin

• interferons

• tissue plasminogen activator

• vaccines

• antibodies

Directed Evolution

mimics natural evolution by imposing stringent selection and screening methodologies to identify proteins with optimized functionality, including genetic diversity, binding, catalytic properties, thermal and environmental stability

Rational Protein Design

• de novo design proteins

• know stuff about its shape + active site and how they fold→ uses detailed knowledge of the structure and function of a protein to make desired changes

• amino acid predictions

• becoming more important

Examples of diagnostic proteins

• enzymes

• antibodies

• biosensors

What is one use of diagnostic proteins?

• Biodetection in pregnancy test

• Certain antigen present in pregnant people → production + binding of antibodies

Examples of enzymes used commercially

Industrial

• food/ textile production

Biotech enzymes

• restriction enzymes

• thermostable polymerases

• ligases

Issues of with purifying natural proteins

• Availability of starting material→ cells not dedicating to making the protein of interest

• Abundance of protein within cell/tissue

• Contamination/ infection of tissue - problem with using natural sources e.g prions - contaminated blood scandal in the news

• Ethical considerations

• Purification considerations

• Cost

General Steps of Recombinant Protein Production

1. Isolate gene from natural host e.g human

2. Engineer Gene so that it can be carried by another host e.g bacteria like E.coli

3. Purify recombinant protein

What are the advantages of recombinant protein production?

• Can use non-pathogenic host that is easily cultured→ bacteria

• Genetic engineering allows high expression of required proteins

• Much reduced contamination and infection risk

• Production may be more ethically acceptable procedure

What is hGH?

• Human growth hormone

• Naturally produced in minute quantities in pituitary gland

• Required for growth and development

• decline in hGH with age

• Purified hGH was used in treatment of growth deficiencies

How was natural hGH produced pre-1985?

• Animal hormone incompatible

• hGH was isolated from human cadavers

• Major HIV and prion issues - natural hGH is now banned!

How is hGH produced now?

• Genentec started to produce it and was approved in 1985

• mainly produced in E. Coli – it is a very simple protein

  • Single polypeptide with just 2 disulphide bonds

  • No cofactors required for activity

  • Not toxic to host cell

  • Stable and soluble

What basic principles does gene cloning use?

• Extraction of nucleic acids→ DNA purification

• Manipulation of DNA/RNA:

  • Type II restriction endonucleases

  • DNA ligases

• Electrophoresis

• PCR

• Reverse transcription

How are recombinant DNA molecules made?

1. EcoR1 produces fragments with ‘sticky’ ends

2. Joined using T4 DNA Ligase

Where does EcoRI cut DNA?

5'...GAATTC...3'

3'...CTTAAG...5'

After creating recombinant DNA, what is the next step?

Amplification

Why is the clonal growth of bacteria useful for DNA amplification?

• Clonal growth

• One bacteria produces two copies of itself

• All bacterial DNA is copied

• Individual clones amplify into colonies of bacteria

• Cloning can refer to the multiple copies of bacteria cells or sequences of DNA produced during clonal growth

Why are the plasmids in bacteria useful for DNA amplification?

• Each bacterium has one copy of its chromosomal DNA

• Plasmids are additional (non-chromosomal) bacterial DNA

• Bacteria also copy plasmids during clonal growth

• Usually have specific or special functions:

  • Antibiotic resistance

  • Nitrogen fixation

  • Virulence

Horizontal Gene Transfer

the transfer of plasmids between bacteria

In what 3 ways can plasmids be transferred between bacteria?

1. Transformation→ take up DNA from the environment

2. Conjugation →bacterial sex, pilli fusion with another bacteria

3. Transduction (phages)→ from viruses

How can the uptake of plasmids be increased/ made competent?

via heat-shock, electroporation or certain chemicals (e.g. CaCl2)→ stress out bacteria

What was the first ‘gene cloning’ experiment?

• University of California, San Francisco, Stanley Cohen and Herbert Boyer 1973

• Inserted frog (xenopus) DNA into bacterial plasmids to create the first transgenic organism

1. Digest both sets of DNA with EcoR1

2. Combine the two DNA fragment sets and ligate

3. Transfer into bacteria

4. Analyse clones of bacteria (colonies) for frog DNA

What comes next after amplifying recombinant DNA?

Identifying/ selecting for it

What was the issue with the first ‘gene cloning’ experiment?

• selection of different outcomes

• Bayer and Cohen started with a plasmid that carried tetracycline-resistance

• Random recombinant DNA outcomes include:

  • Plasmid without ability to replicate in bacteria

  • Plasmid without antibiotic resistance

  • Plasmid without frog DNA

Modern plasmid cloning vector properties

• Origin of Replication

• Resistance/selection/marker gene

• Multiple cloning site

  • With flanking promoters/regulators

• A marker to select for insertion of fragment

What are multiple cloning (polylinker) sites?

• Area with many different restriction enzyme cutting sites→ Allows insertion of DNA

• Usually one site for each RE in the MCS

• Sometimes two of certain RE (one outside MCS)

What do resistance/ selection/ marker genes do?

• Allow identification of correct plasmids

• lacZ, fluorescent proteins, antibiotic resistance

• Often combined in vectors

How can you prevent plasmids from reclosing?

• ore likely for plasmid to reclose→ its closer- intramolecular reaction

• prevent this happening → alkaline phosphatase → take phosphate from the end of the vector→ not 100% efficient

• Self ligation occurs anywhere

What is one way of distinguishing self-ligation from the wanted molecule?

α-complementation

α-complementation

• Mutant, inactive form of β-galactosidase missing N-terminus portion: α-peptide

• This a-peptide is encoded by the LacZ gene

• Return of this α-peptide is called α-complementation

• Functional β-galactosidase is an enzyme that converts the colourless lactose analogue X-Gal to a blue colour

• When DNA is inserted, interrupting the peptide sequence: no blue colonies anymore, only white ones (the ones we want)

After identifying/ selecting for the right bacteria- what is next?

• grow bacteria

• Separate out plasmid DNA from protein DNA

• use gel electrophoresis to see if extracted DNA has the right architecture

What is an alternative cloning strategy?

• Can use RE digests and ligations to make final virus via MCSs

  • Difficult to plan for frameshifts, orientation and other naturally-occurring RE sites during primer and vector design

How can PCR be useful in gene cloning?

• Can amplify gene of interest using PCR

• Amplification → primer design → design primers so that at 5’ end they contain a restriction enzyme sites

• More amplification → molecules with primers, integration of restriction enzyme site into product

• Use restriction enzymes to cut them out → plasmid

Problem

• gene + restriction site sequence

What does TA cloning involve?

• ligation of PCR products into a vector that has T-overhangs

• Many Taq polymerases leave a 3’ A-overhang and we can use this to clone

  • High-fidelity or proofreading versions do not

• Introduce LacZ marker

• Antibiotic resistance genes

What is the issue with TA cloning?

inefficient→ only one basic interacts instead of restriction enzyme

Which cloning method is an improvement on TA cloning?

TOPO cloning

What is TOPO cloning?

uses Topoisomerase I to ligate DNA fragments instead of DNA ligase

• Topoisomerase I recognises (C/T)CCTT-3’ and covalently recombines DNA

• Much faster reaction (5 mins at Room Temp)

• Topoisomerase I is attached to TOPO vector

• Vector 3’ ends in (C/T)CCTT

• Can be used for TA cloning or made directional via altered primers

• Can be subcloned into gateway destination vectors

General procedure of Gene Cloning

• Obtain and alter gene of interest:

  • RE digestion of DNA

  • RE sites with primers

  • Direct cloning of PCR products

• Perform ligation with plasmid vector, transform bacteria (as in the practical!)

• Select bacterial colonies via antibiotic resistance

  • Extract plasmid DNA

Example of a bacterial transformation + expression of protein

Single plasmid vector contains

• Ampicillin resistance (beta- lactamase gene)

• GFP marker (under L-arabinose-dependent promoter)

Considering how bacteria processes RNA, what is the issue with amplifying DNA in this way?

• Bacteria do not have introns in genes

• No splicing post transcription

• Cannot properly express genes in bacteria

Solution:

• Can use mature eukaryotic mRNA (does not have introns) to make DNA (cDNA) via reverse transcription

The Reverse Transcription Reaction

• To remove introns + produce DNA from RNA

• Take a cell, extract mRNA, amplify it → copy to DNA

• use a primer with a poly D → binds to mRNA poly A tail

• Oligo-dT primer used to get all mRNA

• Uses enzymes found in retroviruses:

  • avian myeloblastosis virus (AMV)

  • Moloney murine leukaemia virus (Mo-MLV)

Why is there a need for Codon Optimisation?

• Genetic code is universal but degenerate

• Different organisms show preferences

• Need to re-engineer code to match preferences of HOST organism.

How can we solve the issue of codon optimisation?

• Make DNA synthetically

• substitute every codon for the optimised codon for the organism that we want it to be expressed in

• PCR extension of seed oligonucleotides

• → Clone it into an appropriate vector and DELIVER it

What are some other expression considerations with using bacteria to produce clones genes?

• Degradation→ bacteria will try to remove/ degrade the protein

Solution: Aggregation

• aggregate/ sequester proteins into vesicles so that it doesn’t mess up the machinery of the cell

What are inducible operons?

proteins that can bind to either activate or repress transcription depending on local environment + needs of the cell

Example: lac operon

What does lac operon do?

encodes the genes necessary to acquire and process the lactose from the local environment, which includes the structural genes lacZ, lacY, and lacA. lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.

Why are protein tags useful?

• separate the protein of interest from the rest of the cells

• Insert gene→ next to gene is the tag

• Tags have specific properties→ binds strongly to the substrate

• Incubate proteins with beads with substrate the tag will bind to

• e.g mix with nickel and protein of interest sticks to beads

• Above = affinity chromatography

Examples of protein tags

• His-tag: six histidine (His) residues: nickel binding

• Maltose binding protein (MBP): increased solubility

• Glutathione-S-transferase (GST): improved separation

What is the point of gateway cloning?

• Patented lambda-based site-specific (att) recombination via lambda and E.coli proteins (Int Xis)

• Easier subcloning (transfer between vectors)

• Maintains reading frame and sense

• Allows transfer to different vectors for different applications

• saves generating your own plasmids

• only have to do this process once (into entry clone)

• generates cDNA libraries of ‘entry clones’

How does Gateway Cloning work?

Entry Clones

• aatL are cut to form ‘sticky ends’

Destination Clones

• have attR, which match attL ‘sticky ends’

• ‘LR’ reaction forms attB Expression clone and a by-product with attP and ccdB

  • expression clone is antibiotic resistant

  • by-product cannot grow in bacteria → dies

  • Destination vector contains resistance gene so may still survive

Where are the DNA fragments for gateway cloning from?

• Restriction endonuclease digestion and ligation

• PCR

• cDNA library

How can bacteriophages be used for gene cloning

• Transduction

• Viruses that infect bacteria

• Can contain lots of DNA → Phage life-cycle involves production of very large amounts of phage DNA within bacteria (lytic phase → many copies)

• Produce encapsulated copies of phage DNA

Monoclonal antibody production

• Inject mouse with antigen

• Antigen made using recombinant DNA

• mouse has immune rejection → take these antibodies

Antibody production in phage

Can be done so that every phage carries a distinct HLA variant (human leukocyte antigen)→ expressing different monoclonal antibody

How can you find the phage expressing the antibody against the antigem?

• antigen on surface of bead → phage that binds to antigen sticks

• wash away everything else

= panning

Panning ~ 3-4 x

1. Exposure of bacteriophage to immobilised target of interest

2. Washed + eluted

3. Bacterium infection

4. Amplification

5. Repeated Exposure

6. Washed and Eluted

7. Verify target binding (ELISA Screening)

8. Isolate Antibodies

9. Amplification

Example of getting highly specific monoclonal antibodies that recognise antigen of interest?

• Cancer cells → express antigens they shouldn’t be expressing e.g through mutation

• Antibody that recognises antigen → specific antibody can target this tissue and not any other

• Can chemically couple cytotoxic drug to antibody → specific mechanism

• = immunotherapy