Cloning and expression of eukaryotic genes in bacteria

KBT

Cloning and expression of eukaryotic genes in bacteria

Problems / obstacle to obtain proper expression of eukaryotic genes in bacteria
Eukaryotic genes
Introns, sometimes >50, 10000-100000Ntds Mammalian genes: vast. Haploid = 3B Ntds Screening DNA library very laborious Some genes less known
So, impossible to screen directly

Specific patterns of regulation brain

So, Successful outcome = skill + Intuition + Good luck

Reaching gene via. mRNA:

No non-coding region Tissue expressing large amounts of desired mRNA
RBC: 50 90% polyA cytoplasmc RNA is globin mRNA

Though, only 1 5% of total RNA = mRNA

polyA tail: Affinity chromatography beads with polyT only mRNA can be eluted selectively Purification and size determination Northern blotting

 Reverse transcription (preparation of cDNA) Oligo T primer Hairpin loop (ss-cDNA) Specific ss-endonuclease Expression vector (for regulatory sequences)

 Reaching the gene via proteins When mRNAs are not obtained:
Reverse translation Polysome precipitation

 Reverse translation for synthetic DNA: Noncellular process Low abundance of mRNA, part of mRNA Use as probe in Northern blotting Partial / complete sequence of protein
Deduction of Ntd sequence
PolyNtd synthesis

 Degeneracy : best section of protein Polysome precipitation Separation of ribosome complexes from the tissues Precipitation by specific antibody Extraction of mRNA from protein complex Purification of mRNA cDNA preparation

Synthesis of complete genes Small proteins Economic interest Modified genes Chemical synthesis by Khorana Method