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1. Recombinant Protein Expression in Prokaryotic cells
Fig 1 Recombinant Protein Expression in Prokaryotic
Cells
Q1: What if the soluble expression cannot be
expressed?
A1: Corresponding measures are as follows:
In order to promote soluble expression of protein,
fusion tags can be added to the N or C terminus of the
target protein, commonly including GST and SUMO tags.
However, considering the downstream application of
protein, label removal should be carried out in the later
stage.
Prokaryotic cells (BL21(DE3)) require an inductor,
IPTG, for protein expression after plasmid
transformation. BL21(DE3), as a commonly used
prokaryotic expression cell, which is selected by most
researchers due to its advantages of short cycle and
low cost. The basic flow of recombinant protein
expression in prokaryotic cells is shown in Figure 1.
1. Introduction
2. Q&As for Recombinant Protein Expression
Q2: What are the causes of low protein
expression?
A2: Possible causes:
(1) Before induction, the protein is toxic.
Corresponding measures are as follows:
Control the background expression level:
A. Use tightly controlled plasmids to reduce copy
number.
B. lac-based promoter expression with glucose added.
C. Glucose was selected as the carbon source
medium.
D. based on T7-based promoter expression, plasmid
containing T7 lysozyme was used.
(2) After induction, the protein becomes toxic.
Corresponding measures are as follows:
Control induced expression level:
A. An adjustable promoter is used.
B. Controllable inducible strains were used.
C. Reduce copy number.
D. Suitable virulent protein expression strains were
used.
E. Secretory expression strategy was adopted.
(3) Codon preference. Corresponding measures are as
follows:
A. Optimization of plasmid DNA codons to fit the host
codon system.
B. Codons were used to adjust the strain.
C. Add new substances: Try a new medium; Improve
ventilation condition and avoid soaking.
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Q3: What causes inclusion bodies to form?
A3: Possible causes:

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(1) Incorrect disulfide bond formation.
Corresponding measures are as follows:
A. Secretory expression strategy was adopted.
B. E.coli, which has cytoplasmic oxidative
function, is used.
(2) Incorrect folding. Corresponding measures
are as follows:
A. Co-expressed molecular chaperones.
B. Supplemented with medium containing
compound chaperones and cofactors.
C. Remove the inducer and add fresh medium:
Low temperature induced expression; Adjust
the inducer concentration.
(3) Low soluble protein is formed.
Corresponding measures are as follows:
A. Change the fusion chaperone protein and
promote the expression of soluble protein.
(4) Post-translational embellishment is crucial.
Corresponding measures are as follows:
A. Changes in the expression host, such as
prokaryotic changes in the eukaryotic
expression system.
3. Characteristics of prokaryotic expression
systems
(1) Clear genetic background.
(2) Rapid propagation.
(3) Low cost.
(4) High expression.
(5) Easy purification of expression products.
(6) Good stability.
(7) Strong anti-pollution ability.
(8) Wide application range.
Q4: What's the reason for the lack of activity?
A4: Possible causes:
(1) Incorrect folding. Corresponding measures are as
follows:
A. Low temperature induced expression.
B. Monitor disulfide bond formation and in vitro folding.
(2) cDNA mutation. Corresponding measures are as
follows:
C. Sequencing before and after plasmid induction.
D. The recA− strain was used to ensure plasmid host
stability.
E. Changes in the expression host, such as prokaryotic
changes in the eukaryotic expression system.