Modified ‘one amino acid-one codon’ engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase

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) P( R ) 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

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

<p>Abstract</p> <p>Background</p> <p>We previously defined a family of restriction endonucleases (REases) from <it>Thermus </it>sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.</p> <p>Results</p> <p>TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the <it>Thermus </it>sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.</p> <p>Conclusions</p> <p>TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.</p

Novel engineered TRAIL‐based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance

Targeting of the TRAIL‐DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD‐O51.4, which is a TRAIL equipped with positively charged VEGFA‐derived effector peptides. The study was performed in multiple cancer cell line‐ and patient‐derived xenografts. A pharmacokinetic profile was established in monkeys. AD‐O51.4 strongly inhibits tumor growth, even leading to complete long‐term tumor remission. Neither mice nor monkeys treated with AD‐O51.4 demonstrate symptoms of drug toxicity. AD‐O51.4 exhibits a satisfactory half‐life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD‐O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD‐O51.4‐driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL‐sensitive and TRAIL‐resistant cancer cells, respectively. The FADD‐dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD‐O51.4 is capable of bypassing the refractoriness of TRAIL. AD‐O51.4‐driven cell death, which exceeds TRAIL activity, is achieved due to the N‐terminally fused polypeptide, containing VEGFA‐derived effector peptides. The high anticancer efficiency of AD‐O51.4 combined with its safety has led to the entry of AD‐O51.4 into toxicological studies