Optimizing assembly and production of native bispecific antibodies by codon de-optimization

When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. κλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a κλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the κλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.     

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.     

Prediction of Directional Changes of Influenza A Virus Genome Sequences with Emphasis on Pandemic H1N1/09 as a Model Case

Influenza virus poses a significant threat to public health, as exemplified by the recent introduction of the new pandemic strain H1N1/09 into human populations. Pandemics have been initiated by the occurrence of novel changes in animal sources that eventually adapt to human. One important issue in studies of viral genomes, particularly those of influenza virus, is to predict possible changes in genomic sequence that will become hazardous. We previously established a clustering method termed ‘BLSOM’ (batch-learning self-organizing map) that does not depend on sequence alignment and can characterize and compare even 1 million genomic sequences in one run. Strategies for comparing a vast number of genomic sequences simultaneously become increasingly important in genome studies because of remarkable progresses in nucleotide sequencing. In this study, we have constructed BLSOMs based on the oligonucleotide and codon composition of all influenza A viral strains available. Without prior information with regard to their hosts, sequences derived from strains isolated from avian or human sources were successfully clustered according to the hosts. Notably, the pandemic H1N1/09 strains have oligonucleotide and codon compositions that are clearly different from those of human seasonal influenza A strains. This enables us to infer future directional changes in the influenza A viral genome.     

Nucleotide composition-based prediction of gene expression efficacy

A correlation between efficacy of gene expression and nucleotide composition of its protein-coding sequences was studied. It was found that measures based exclusively on codon frequencies are analogous to codon adaptation index [1] and do not adequately reveal this correlation. A more general measure is suggested, i.e., elongation efficacy index, which takes into account both the codon frequencies and the level of local mRNA complementarity. Utilization of this measure facilitated adequate recognition of highly expressed genes in 18 unicellular organisms, i.e., 14 eubacteria, three archaebacteria, and yeast.     

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the “hemoglobin (Hb) superfamily.” The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His¹¹⁷ and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His¹¹⁷ is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.     

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control

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Analysis of donor splice sites in different eukaryotic organisms

We present here a new algorithm for functional site analysis. It is based on four main assumptions: each variation of nucleotide composition makes a different contribution to the overall binding free energy of interaction between a functional site and another molecule; nonfunctioning site-like regions (pseudosites) are absent or rare in genomes; there may be errors in the sample of sites; and nucleotides of different site positions are considered to be mutually dependent. In this algorithm, the site set is divided into subsets, each described by a certain consensus. Donor splice sites of the human protein-coding genes were analyzed. Comparing the results with other methods of donor splice site prediction has demonstrated a more accurate prediction of consensus sequences AG/GU(A,G), G/GUnAG, /GU(A,G)AG, /GU(A,G)nGU, and G/GUA than is achieved by weight matrix and consensus (A,C)AG/GU(A,G)AGU with mismatches. The probability of the first type error, E1, for the obtained consensus set was about 0.05, and the probability of the second type error, E2, was 0.15. The analysis demonstrated that accuracy of the functional site prediction could be improved if one takes into account correlations between the site positions. The accuracy of prediction by using human consensus sequences was tested on sequences from different organisms. Some differences in consensus sequences for the plant Arabidopsis sp., the invertebrate Caenorhabditis sp., and the fungus Aspergillus sp. were revealed. For the yeast Saccharomyces sp. only one conservative consensus, /GUA(U,A,C)G(U,A,C), was revealed (E1 = 0.03, E2 = 0.03). Yeast is a very interesting model to use for analysis of molecular mechanisms of splicing. Received: 14 October 1996 / Accepted: 30 January 1997