Recombinant DNA technology

Recombinant DNA combines sequences that do not occur together in nature. Heterologous expression produces a protein in a host unlike its natural source, for example a human enzyme in Escherichia coli.

The workflow

1. Obtain the gene: amplify it by PCR from genomic DNA or from cDNA (reverse-transcribed mRNA, which lacks introns that bacteria cannot splice), or order a synthetic gene with codons optimised for the host. 2. Clone it into a vector. Plasmids are circular, self-replicating DNA molecules carrying an origin of replication, a selectable marker (usually antibiotic resistance) and a multiple cloning site. Expression vectors add a promoter, ribosome binding site, start and stop signals, a terminator and often an affinity-tag sequence. 3. Transform host cells by heat shock (chemically competent cells) or electroporation. 4. Select transformants on antibiotic plates; screen colonies by colony PCR, restriction digest or sequencing. 5. Grow, induce, lyse and purify.

Restriction enzymes and ligation

Type II restriction endonucleases recognise short, usually palindromic sites and cut within them. EcoRI cuts G^AATTC to leave 5′ overhangs (sticky ends); others leave 3′ overhangs or blunt ends. Cutting vector and insert with the same enzyme gives complementary sticky ends; using two different enzymes gives directional cloning and stops the vector re-closing on itself. T4 DNA ligase seals the backbone nicks, forming phosphodiester bonds using ATP. Modern alternatives include Gibson assembly (overlapping ends joined by exonuclease, polymerase and ligase) and Golden Gate assembly using type IIS enzymes that cut outside their recognition site.

Controlled expression

In the widely used T7 system, the gene sits behind a T7 promoter, and the host strain carries the T7 RNA polymerase gene under a lac-controlled promoter. The Lac repressor blocks expression until IPTG, a non-hydrolysable mimic of allolactose, is added; it binds the repressor and releases it from the operator. Delaying induction until cells are dense limits toxicity and maximises yield.

Common problems

• Inclusion bodies: insoluble aggregates of misfolded protein; mitigated by lower temperature, weaker induction, solubility tags or co-expressed chaperones, or by refolding.
• Codon bias: rare codons in the host stall ribosomes; fixed by codon optimisation or strains supplying rare tRNAs.
• Missing post-translational modifications and disulfides: bacteria do not glycosylate like human cells, so yeast, insect or mammalian (e.g. CHO) cells are used for many therapeutic proteins.

Site-directed mutagenesis

To change one residue, the plasmid is amplified with a pair of complementary primers carrying the mismatch. The parental template, isolated from E. coli and therefore methylated, is destroyed with DpnI, which cuts only methylated GATC sites, leaving the newly synthesised mutant plasmid. Comparing wild-type and mutant enzymes tests which residues matter for catalysis, binding or stability.