QPSOBT: One codon usage optimization software for protein heterologous expression
ABSTRACT QPSOBT is a codon usage optimization software based on the Quantum-behaved Particle Swarm Optimization (QPSO) algorithm. It can design synthetic genes of multikilobase sequences for protein heterologous expression rapidly. The program runs on .NET platform. Compared to the existing codon optimization software and web services, QPSOBT is able to generate better results when DNA/RNA sequence length is less than 6 kb which is a commonly-used range, especially when some restriction sites need to be removed. QPSOBT is freely available (www.sigcib.org/qpsobt.html).
Full-textDOI: · Available from: Yujie Cai, Oct 08, 2014
- SourceAvailable from: ncbi.nlm.nih.gov
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- "However, due to the large and discrete search space which contains all possible DNA sequences that can encode the target protein, solving the MILP using these methods may require a long computational time. Thus, alternative methods, such as GASCO  and QPSOBT , have been proposed for solving ICO using genetic algorithm and particle swarm optimization. Although these heuristic methods are more efficient than conventional MILP solving procedures, they still require a significant amount of computational resources due to the iterative nature of the algorithms. "
ABSTRACT: Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology.BMC Systems Biology 10/2012; 6(1):134. DOI:10.1186/1752-0509-6-134 · 2.44 Impact Factor
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- "These techniques are often based on previously identified preferred codons, relative to the system of interest. During the process of optimization, existent codons are replaced by a set of more suitable host codons [6, 13–15]. Because the gene of interest, cysC, is a mammalian gene that is poorly expressed in most bacterial systems, several alternatives exist to improve gene expression. "
ABSTRACT: Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the human cystatin C (cysC) gene using the preferred codons of the prokaryotic system may significantly increase cysC expression in Escherichia coli (E. coli). Specifically, cysC expression may be increased by removing unstable sequences and optimizing GC content. According to E. coli expression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimized cysC (co-cysC) and wild-type cysC (wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid into E. coli BL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni(2+)-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase in cysC expression in E. coli expression produced by codon optimization techniques may be applicable to commercial production systems.BioMed Research International 10/2012; 2012:732017. DOI:10.1155/2012/732017 · 2.71 Impact Factor
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ABSTRACT: Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5' thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ∼3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ∼4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ∼3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.PLoS ONE 11/2010; 5(11):e15056. DOI:10.1371/journal.pone.0015056 · 3.23 Impact Factor