High-level expression of tamavidin 2 in human cells by codon-usage optimization

Takakura, Yoshimitsu; Katayama, Sakurako; Nagata, Yuki

doi:10.1080/09168451.2014.991690

Cited by 6 publications

(2 citation statements)

References 31 publications

(46 reference statements)

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“…7,13,27,28,[30][31][32]43 We first attempted to re-establish a more balanced expression of the two light chains by optimizing the sequence encoding the less expressed kappa light chain, but this approach did not improve the situation. We then considered the opposite approach and tested the possibility of reducing the expression of the lambda light chain by incorporation into the coding sequence different degrees of less favorable codons for expression in mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

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

Magistrelli

Poitevin

Schlosser

et al. 2016

mAbs

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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. kλ 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 kλ 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 kλ 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.

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Section: Discussionmentioning

confidence: 99%

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

Magistrelli

Poitevin

Schlosser

et al. 2016

mAbs

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“…Codon harmonization is important for the expression of foreign gene. For example, codon-usage modification resulted in higher avidin expression in E. coli [43] and P. pastries [44,45]. C. glutamicum belongs to the high G+C Gram-positive Actinobacteria, and C. glutamicum metK had 56.1% G+C content, whereas SAM2 had only 41.9% G+C content and may contain codons that are rarely used in C. glutamicum.…”

Section: Codon-optimized Sam2 Sam2-c Could Be Overexpressed and Itsmentioning

confidence: 99%

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

Han

Wang

2015

Biotech and App Biochem

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Two genes encoding methionine adenosyltransferase, SAM2 from Saccharomyces cerevisiae and metK from Corynebacterium glutamicum, were individually cloned into pDXW-8, the shuttle vector between Escherichia coli and C. glutamicum, and overexpressed in E. coli DH5α and C. glutamicum ATCC13032. In DH5α, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032, metK was overexpressed, its product MetK showed the enzyme activity and could convert l-methionine to S-adenosyl-l-methionine (SAM). However, when SAM2 was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM from l-methionine was observed. Reverse transcription PCR analysis and SDS-PAGE showed that SAM2 was transcribed but not translated in C. glutamicum. Therefore, SAM2-C, a mutant SAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convert l-methionine to SAM. Finally, SAM2-C and metK were individually overexpressed in E. coli BL21(DE3), and their products SAM2-C and MetK were purified and characterized. The optimum activity for both enzymes was found at pH 8.5 and 35 °C; SAM2-C and MetK have similar K for ATP, but quite different K for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.

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Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment

Liu

Liu²,

Liu

2020

New Biotechnology

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High-level expression of tamavidin 2 in human cells by codon-usage optimization

Cited by 6 publications

References 31 publications

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

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

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment

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