Method of posttranslational modification by adding mycrosomal membrane in cell-free protein synthesis
A protein synthesis, cell-free technology, applied in animal/human proteins, chemical instruments and methods, cytokines/lymphokines/interferons, etc., can solve the problems of lack of versatility
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[0101] In the method for preparing an extract proposed by the present inventors above, there are no particular limitations on other steps as long as at least the step of rapid freezing is included. For example, it can be obtained from Escherichia coli or wheat germ, etc. Various methods that have been used in the extraction of cell line protein synthesis are used to crush insect cultured cells and extract. Among them, it is preferable to disrupt the cultured insect cells by rapidly freezing the cultured insect cells, thawing, and centrifuging.
[0102] After thawing the above-mentioned rapidly frozen cultured insect cells, when performing centrifugation, thawing can be achieved by, for example, thawing in a water bath or an ice-water bath at -10°C to 20°C, standing at room temperature (25°C), etc. , but in order to prevent the inactivation of the components necessary for protein synthesis and reliably prevent the reduction of protein synthesis ability, it is preferably thawed...
Embodiment 1
[0178] (1) Preparation of expression plasmid
[0179] In the post-translational modification, sugar chain modification (N-glycosylation) was used as a model, and pro-TNF-GLC (an N-glycosylation site artificially introduced into the mature region of the protein) known to produce sugar chain modification was used. mutant human antitumor factor), the in vitro synthesis of this glycoprotein was attempted. figure 1 Schematic representation of the constructed expression plasmids. In addition, the nucleotide sequence of the polyhedrin 5'-UTR used is shown as SEQ ID NO: 1, and the entire nucleotide sequence of the pro-TNF-GLC gene is shown as SEQ ID NO: 2.
[0180] The expression plasmid was constructed by pT N T vector (manufactured by Promega) was used as a template, and PCR primers having a BamHI site added to the ends of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 were used to carry out self-ligation (selfligation) after BamHI digestion. A BamHI site was int...
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