Why are eukaryotic genes difficult to express in E. coli?
Eukaryotic gene expression in bacterial system Escherichia coli faces multiple barriers collectively contributing to failure. Bacterial RNA polymerase cannot splice introns as bacteria lack spliceosome, producing nonfunctional transcripts. Post-translational modification systems for glycosylation, phosphorylation, and formation of correct disulfide bonds in endoplasmic reticulum are absent. Cytoplasmic milieu, chaperone repertoire, and codon usage diverge significantly, causing improper folding, codon bias mediated stalling, and proteolysis. Consequently, most complex eukaryotic proteins aggre
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.