title: Modified 'one amino acid-one codon' engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase.
abstract: BACKGROUND
  An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host's viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host's coding plasmid replication.TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM).
  
  
  RESULTS
  We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) PR promoter. Codon usage based on a modified 'one amino acid-one codon' strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields 'codon randomization' strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high 'toxicity' of the REase-MTase protein.
  
  
  CONCLUSIONS
  Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified 'one amino acid-one codon' method tuned for thermophile-coded genes was applied to obtain overexpression of the 'toxic' taqIIRM gene. The method appears suited for industrial production of thermostable 'toxic' enzymes in E. coli. This novel variant of the method biased toward increasing a gene's AT content may provide economic benefits for industrial applications.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.forename: Olga
authors.initials: O
authors.lastname: Zolnierkiewicz
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Sliwinska
authors.forename: Joanna
authors.initials: J
authors.lastname: Jezewska-Frackowiak
authors.affiliation: Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. piotr.skowron@ug.edu.pl.
authors.forename: Piotr M
authors.initials: PM
authors.lastname: Skowron
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013824:Thermus / Q000201:enzymology*
chemical_list: D003062:Codon; D012333:RNA, Messenger; D011994:Recombinant Proteins; C042367:endodeoxyribonuclease TaqII; D015252:Deoxyribonucleases, Type II Site-Specific