2. Introduction
A method in which a cloned gene is inserted into a suitable vector and introduced into
Escherichia coli for expression of a large amount of protein is generally referred to as
prokaryotic expression. This method has applications in protein purification,
localization and functional analysis. E. coli used to express recombinant proteins has
the following characteristics: easy to grow and control; materials used for bacterial
culture are less expensive than mammalian cell systems; A wide variety of E. coli strains
and matching plasmids with various properties are available. However, proteins
expressed in E. coli often form inclusion bodies to affect the biological activity and
conformation of the expressed protein due to lack of modification and post-
translational processing such as glycosylation and phosphorylation.
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3. Introduction
Expression vectors play an important role in genetic engineering. Prokaryotic
expression vectors are usually plasmids. Typical expression vectors should have the
following components:
(1) Selecting a coding sequence of the marker;
(2) A promoter capable of transcribed;
(3) Transcriptional regulatory sequences (transcriptional terminator, ribosome binding
site);
(4) A multi-restriction cleavage site linker;
(5) Sequences autonomously replicated in the host.
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4. Introduction
The general procedure for prokaryotic expression is as follows: obtaining the gene of
interest - preparing the expression vector - inserting the gene of interest into the
expression vector (sequencing verification) - transforming the expression host strain -
inducing expression of the target protein - analysis of the expressed protein -
amplification, purification, further detection.
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5. Primary reagent
1. LB medium.
2. 100 mM IPTG (isopropylthio-β-D-galactoside): 2.38 g of IPTG was dissolved in 100 ml
of ddH2O, filtered through a 0.22 μm filter, and stored at -20 °C.
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6. Experimental procedure
(1) Obtaining the gene of interest
(2) Construction of recombinant expression vector
(3) Obtaining the expression strain containing the recombinant expression
plasmid
(4) Induced expression
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7. Precautions
1. When selecting an expression vector, it should be considered according to the final
application of the expressed protein. For ease of purification, fusion expression can be
selected; if a native protein is obtained, non-fusion expression can be selected.
2. In the fusion expression, when the foreign DNA is selected and linked to the carrier
molecule, the reading of the cryptographic structure during transcription and
translation cannot be interfered.
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