Recombinant DNA technologies

How is rDNA made

Cutting: restriction enzymes

Pasting: Ligase

How are restriction enzymes be used for cloning

• Recognise palindromic sequences aka restriction sites

• restriction sites for cloning usually are 4 or 6 nucleotides

How do restriction enzymes cut

Cut both DNA strands creating sticky or blunt ends

Palindrome

A-T

C-G

C,D,F

Agarose gel electrophoresis

Load on agrose gel

Run with some voltage

stain with fluorescent dye and visualise

UV light

Size can be calculated from position of known molecular weight marker bands

DNA - negatively charged

Small= moves quickly

What is DNA ligase

Used for sticking back together: repair and replication

ATP dependent enzyme that links DNA strands

Ligates compatible sticky ends (more efficient) and blunt ends

What are selection markers

Cells that have the plasmid

Genes for antibiotic resistance or growth on specific media

What do plasmids need for cloning

Selection marker

Region where DNA can be inserted

Cloning workflow: Vector preparation

Restrcition digest at 27 dgerees

purify

Insert prepation

PCR 2-3 HOURS

Restriction digest at 37

purfication

Ligation

T4 DNA Ligase at 20 degrees

Optional purfication

Transformation

Prepare compentent cells

transformation of cells and plating

Cloning screening

Blue-white screening

restriction digest colony PCR sequencing

• Insert ligates into vector

• Vector may also ligate back onto itself

What is blue-white screening

A plasmid that contains LacZ gene, which encodes the enzyme b-galactosidase

If LacZ gene intact = Blue dye , B-gal active

If DNA insert disrupts LacZ gene= white, B-gal inactive

What properties do we want for hosts of cloning and expression

Grows rapidly in inexpensive medium

Non pathogenic

Generally stable

Has many tools for genetic manipulation

Allow high level of expression of genes

Difference between hosts and clones

Hosts for cloning and expression are not the same

What are some hosts for cloning and expression

Where does protein synthesis begin in the cell?

Any enzyme that needs to be secreted with come from endoplasmic reticulum

Protein synthesis begins in the Endoplasmic Reticulum (ER), where ribosomes translate mRNA into a preprohormone.

What is a "preprohormone"?

A preprohormone is the initial, inactive form of a hormone that includes a signal sequence directing it to the ER. The signal sequence is cleaved off, transforming it into a prohormone.

How is the prohormone processed in the cell?

The prohormone is packaged into transport vesicles and sent to the Golgi complex, where it undergoes further modification and becomes an active hormone.

What role does the Golgi complex play in hormone activation?

How is recombinant insulin made

Short peptides such as insulin not stable

peptides stablaised by fusion into large protein

Sequence of insulin can be modified

How can we module insulin-release profile

Mix with protein to slow release

Introduce amino acid changes

Chemical modification

Describe the process of insulin synthesis.

• Preproinsulin (inactive form) has a signal sequence and three chains (A, B, C).

• The signal sequence is removed, creating proinsulin.

• In the Golgi, the C chain is removed, leaving only the A and B chains connected by disulfide bonds, forming active insulin.

What are the A and B chains in insulin?

The A chain has 21 amino acids, and the B chain has 30 amino acids. They are connected by disulfide bonds, forming the structure of active insulin.

Release profile of insulin

Lispro

Reversal of Lys/Pro in B chain

Forms dimers very ineffciently

rapid release

Glargine

Lantus

One deletion and 3 additions

PI: Isoelectric point

Slow release

PI value

proteins around PI value tend to precipitate

Detemere

Thr30 deleted

fatty acid is added on Lys29

causes it to bind very well to albumin

Slow release

What is Factor VIII

• Blood clotting factor

• Used for treatment of haemophilia

• Very large protein

• Glycosylated- can’t be made in bacteria

Cloning of factor VIII

Contains several introns

requires copies to be made from mRNA

Plasmid with gene used to transfect mammalian cells

Plasmid integrates with genome: copies amplified and cell line with highest number of copies used for production

How can Factor VIII be made

Batch culture

Continuous culture

purified from culture medium