Advanced genetic strategies for recombinant protein expression in Escherichia coli.
Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.
Sørensen
Hans Peter
HP
Laboratory of BioDesign, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, Denmark.
Mortensen
Kim Kusk
KK
D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins