Design, synthesis and expression of a human interteukin-2 gene incorporating the codon usage bias found in highly expressed Escherichia coli genes

Williams, Daniel W.; Regier, Dean A.; Akiyoshi, Donna E.; Genbauffe, F S; Murphy, J R

doi:10.1093/nar/16.22.10453

Cited by 64 publications

(18 citation statements)

References 28 publications

(18 reference statements)

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“…125 I was obtained from NEN Life Science Products. Wild-type IL-2 and 2D1 (L18M/L19S) were previously prepared via cassette mutagenesis on a synthetic IL-2 gene (36,37). Proteins were expressed in E. coli, refolded, and purified as described previously (38).…”

Section: Methodsmentioning

confidence: 99%

Increased Endosomal Sorting of Ligand to Recycling Enhances Potency of an Interleukin-2 Analog

Fallon¹,

Liparoto²,

Lee³

et al. 2000

Journal of Biological Chemistry

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An interleukin-2 (IL-2) variant containing adjacent point mutations (L18M/L19S, termed 2D1) displaying binding affinity to the heterotrimeric IL-2 receptor similar to that of wild-type IL-2 (WT) had been previously found to surprisingly exhibit increased bioactivity in a peripheral blood lymphocyte proliferation assay. In order to provide an explanatory mechanism for this unexpected potency enhancement, we hypothesize that altered endocytic trafficking of the 2D1 variant might be responsible by increasing the number of ligand-receptor complexes. We demonstrate here that the internalization kinetics of 2D1 via the high affinity IL-2 receptor are equivalent to those of WT but that a significantly increased fraction of internalized 2D1 is sorted to recycling instead of to lysosomal degradation. We further find a reduced pH sensitivity of binding to IL-2 receptor ␣ relative to IL-2 receptor ␤ compared with WT, which could be responsible for the altered sorting behavior of 2D1 in the acidic endosomal compartment. Accordingly, the 2D1 variant displays a half-life 36 h longer than that of IL-2 in T-lymphocyte culture at concentrations equal to the K D of the IL-2 receptor. The extended half-life of intact 2D1 provides enhanced mitogenesis as compared with IL-2. In addition, 2D1 stimulates natural killer cells to a lesser degree than IL-2 at equal concentrations. We conclude that this IL-2 variant provides increased mitogenic stimulation that could not be easily predicted from its cell surface receptor binding affinity while minimizing undesired stimulation of natural killer cells. This concept of altering trafficking dynamics may offer a generalizable approach to generating improvements in the pharmacological efficacy of therapeutic cytokines.

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Section: Methodsmentioning

confidence: 99%

Increased Endosomal Sorting of Ligand to Recycling Enhances Potency of an Interleukin-2 Analog

Fallon¹,

Liparoto²,

Lee³

et al. 2000

Journal of Biological Chemistry

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“…The mutant IL-2 library was prepared by cassette mutagenesis on a synthetic IL-2 gene under the control of an IPTG-inducible trcpromoter (Williams et al, 1988). A 93-mer oligonucleotide template corresponding to the coding strand of the mutant cassette was synthesized.…”

Section: Methodsmentioning

confidence: 99%

Mutagenic Analysis of a Receptor Contact Site on Interleukin-2: Preparation of an IL-2 Analog with Increased Potency

Berndt¹,

Chang²,

Smith³

et al. 1994

Biochemistry

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Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells. Combinatorial cassette mutagenesis was used to investigate the functional role of a continuous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor. A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-2-dependent cell line. From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified and further characterized by biological and receptor binding assays. Viewed in the context of the recently revised 2.5-A crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons. Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutions at this position, even a conservative Leu-->Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting in a significant loss of function. In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.

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“…Because low-dose immunization of rBCG has been suggested to act as a prophylactic vaccination against HIV-1 (15, 28), we adopted the preferred codon of BCG to enhance the expression of the foreign HIV gene. In recombinant protein production, the potency of codon-optimized gene expression systems was demonstrated in Escherichia coli (39) and in mammalian cells (42). These results clearly show that codon-optimized recombinant genes induce vigorous expression by foreign genes in the host.…”

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confidence: 99%

Mycobacterial Codon Optimization Enhances Antigen Expression and Virus-Specific Immune Responses in RecombinantMycobacterium bovisBacille Calmette-Guérin Expressing Human Immunodeficiency Virus Type 1 Gag

Kanekiyo

Matsuo

Hamatake

et al. 2005

J Virol

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Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.

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Design, synthesis and expression of a human interteukin-2 gene incorporating the codon usage bias found in highly expressed Escherichia coli genes

Cited by 64 publications

References 28 publications

Increased Endosomal Sorting of Ligand to Recycling Enhances Potency of an Interleukin-2 Analog

Increased Endosomal Sorting of Ligand to Recycling Enhances Potency of an Interleukin-2 Analog

Mutagenic Analysis of a Receptor Contact Site on Interleukin-2: Preparation of an IL-2 Analog with Increased Potency

Mycobacterial Codon Optimization Enhances Antigen Expression and Virus-Specific Immune Responses in RecombinantMycobacterium bovisBacille Calmette-Guérin Expressing Human Immunodeficiency Virus Type 1 Gag

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