Agenesis methods, such as UV irradiation, chemical mutagens, and random transposon
Agenesis methods, such as UV irradiation, chemical mutagens, and random transposon mutagenesis. A very useful tool for studying the effect of gene knockouts on FPS-ZM1 chemical information recombinant protein expression and other properties/phenotypes is the Keio collection, a publicly available library of all single knockouts of all the non-essential E. coli K-12 genes [54]. In addition to the classical mutagenesis strategies, new techniques for genome engineering have been PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 developed recently for generating libraries in which the expression of chromosomally encoded genes can been up- or downregulated. These techniques include global transcription machinery engineering (gTME) [55] and trackable multiplex recombineering (TRMR) [56]. These and other genome engineering technologies may be employed to access phenotypes that may be difficult to obtain via classical mutagenesis approaches [57].2.1 Strain engineering by classical mutagenesisOne of the most frequently encountered phenotypic consequences of recombinant protein expression is growth retardation or complete growth arrest of the host following induction of gene overexpression. More than a decade ago, Walker and coworkers isolated E. coli BL21(DE3) mutant strains carrying spontaneously acquired suppressor mutations that alleviate the toxicity caused by the production of cytotoxic proteins under the control of the strong T7 promoter [58]. These strains, which are called C41 and C43 or “Walker strains”, are widely PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 used to produce increased levels of hard-to-express proteins primarily because they allow increased biomass production. Not surprisingly, it was later found that the mutations in these strains reduce the translational efficiency of the T7 RNA polymerase [59]. C41 and C43 are currently commercially available by Avidis. Recently, Bowie and co-workers used a combination of the mutagenic base analog 2-aminopurine and the mutator gene mutD5 (a mutated dnaQ gene causing a DNA proofreading defect), to evolve E. coli strains which accumulate markedly enhanced amounts of a variety of different Mycobacterium tuberculosis rhomboid family proteins and other prokaryotic and eukaryotic integral membrane proteins [60]. These strains were found to produce up to 90-fold higher amounts of protein compared to the parental strain TOP10. In an analogous manner, our group has used the chemical mutagen N-methyl-N’-nitro-N-nitrosoguanidine to generate E. coli mutants that confer up to 5-fold greater yields of properly assembled full-length IgG antibodies in the bacterial periplasm [61]. In another example of classical strain mutagenesis for enhanced recombinant protein production, Skretas and Georgiou usedinsertional mutagenesis of the Tn5 transposon together with fluorescence-activated cell sorting (FACS), to isolate E. coli MC4100A variants that accumulate increased amounts of the membrane-inserted human GPCR central cannabinoid receptor (CB1) [62]. Genes, gene fragments or operon fragments that favorably affect protein expression can be isolated from plasmid libraries co-expressing genomic fragments. Alternatively, individual intact genes can be identified using the ASKA library, an ordered library of all the E. coli ORFs transcribed from the inducible T5lac promoter [63]. Using this library, our group identified E. coli proteins that enhanced the yields of the membrane-embedded form of the human GPCR bradykinin receptor 2 (BR2) [64]. One of these, the putative DNAbinding protein of unknown function YbaB, conferred a 10-fol.
