Methods and systems for autoinduction of protein expression

An inducible and purposeful technology, applied in the field of self-induced protein expression and system, can solve the problem of expensive preparation

Active Publication Date: 2018-01-02
ADAGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this system requires the growth of E. coli transformants in specialized C.R.A.P. phosphate-limiting media, which are quite expensive to prepare

Method used

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  • Methods and systems for autoinduction of protein expression
  • Methods and systems for autoinduction of protein expression
  • Methods and systems for autoinduction of protein expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1: Construction of plasmid DPA1

[0082] The first plasmid, DPA1 (dual-plasmid autoinduction 1 ), was based on the multi-copy pBluscript KS(+) backbone, and the promoter used for protein expression was the lac promoter (SEQ ID NO. 3) lacking any lacI ORF sequence. The shorter lac promoter is fully functional as it is fully repressed by the lad repressor and induced by inducers such as lactose or IPTG. The target gene can be cloned into a multiple cloning site (NdeI and XbaI sites are used here as examples) downstream of the lac promoter. To reduce basal or leaky expression, the lac promoter was preceded by a strong tHP transcription terminator (SEQ ID NO. 2). In addition, a lambda terminator (SEQ ID NO. 4), another strong transcriptional terminator, was placed downstream of the target gene to further reduce leaky expression.

[0083] The DPA1 plasmid map is shown in figure 2 middle. The complete sequence is shown in SEQ ID NO.1.

[0084]The size of plas...

Embodiment 2

[0086] Embodiment 2: Construction of plasmid pLysS MCS

[0087] Plasmid pLysS MCS was constructed to allow insertion of multiple DNA fragments into vector pLysS. We chose the Tet ORF in the vector pLysS as the insertion site for the MCS (Multiple Cloning Site), because the Tet ORF has been disrupted by the gene encoding T7 lysozyme and the tetracycline resistance gene is no longer effective. In order to make the size of the plasmid smaller and easy to handle, we removed the DNA fragment (715bp) from the unique SphI site to the stop codon in the Tet ORF, and replaced it with a DNA fragment containing a multiple cloning site, whose DNA sequence as follows:

[0088] SEQ ID NO.5:

[0089]

[0090] The plasmid map of pLysS MCS is shown in Figure 3A middle. The sequence is SEQ ID NO.6.

Embodiment 3

[0091] Embodiment 3: Construction of DPA2

[0092] lacI q Inserted between the unique KpnI and AscI sites within the multiple cloning site of pLysS MCS to construct plasmid DPA2 ( two-plasmid autoinduction 2). lacI q The transcription of T7 lysozyme is opposite to that of T7 lysozyme. The plasmid map of DPA2 is shown in Figure 3B middle. The sequence is SEQ ID NO.7. DPA2 contains a cryptic promoter that may be constitutive. LacI q Expressed from DPA2 and is functional because in the absence of DPA2, DPA1 cannot be converted to not have lacI in the F' factor q bacterial strains (data not shown).

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Abstract

Methods and systems for autoinduction of gene expression, without the need to add exogenous inducers. A dual genetic element system, which includes a first, high copy number genetic element comprisinga first gene of interest that is under the control of an inducible promoter, and a second, low copy number genetic element comprising a gene encoding a transcriptional factor which, upon expression,regulates transcription from the inducible promoter, wherein activation of transcription from the inducible promoter does not require addition of an exogenous inducer.

Description

technical field [0001] The present disclosure generally relates to methods and systems for in vitro gene expression, particularly genetic elements such as plasmids with regulatory elements. Background technique [0002] During antibody screening processes such as phage display, typically hundreds or even thousands of hits are screened to identify various antibody fragments that bind multiple epitopes of a given target. For each of these hits it was necessary to prepare sufficient quantities of expressed and purified antibody fragments for further characterization. Typically, the genes encoding the corresponding antibody fragments are subcloned together into different expression vectors, or a phage display vector carrying the gene of interest is transformed into an expression vector. Then, the expression vector is transformed into a host cell (typically, Escherichia coli), and the transformant is inoculated into a small culture (1-3 ml) for overnight growth. When the cultur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/79C12N15/63C12N15/64
CPCC12N15/63C12N15/635C12N15/70C07K16/06C07K2317/14C07K2317/55C12N15/72C12N15/73
Inventor 杜方勇罗培志
Owner ADAGENE INC
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