A: There is a risk of not expressing recombinant proteins, which may be related to the properties of the protein itself, such as membrane adhesion proteins, plant derived proteins, or proteins with excessive or insufficient molecular weight, toxic proteins, etc.

A: You can try from the following aspects.

a、Optimize host cells（Origami B(DE3)，Rosetta gami(DE3)，Shuffle）.

b、Co expression with molecular chaperones can help proteins form the correct three-dimensional structure.

C、Fusion tag, (SUMO/GST/MBP/etc)（This is our most commonly used method, with the most commonly used label being sumo, and the prokaryotic system providing free enzyme digestion）.

d、Replace the expression vector. The pET series vector has a high expression level, but also a high probability of inclusion bodies. The pCold low-temperature expression vector theoretically can improve protein solubility.

e、Optimize cultivation conditions (temperature, IPTG concentration, medium composition).

f、Secretory expression, fusion of E. coli secreted signal peptides, guiding proteins to the periplasmic space (not recommended, periplasmic space protein quantity is low, concentration is low).

A: Perhaps there is, theoretically it may not have as good activity as the supernatant protein.

A: Affinity purification is preferred. If gel filtration chromatography, ion exchange chromatography, etc. are required, additional charges are required.

A: HIS/GST/MBP/Flag/Fc.The cost of Flag filler is relatively high and requires additional charges.

A: sumo、TEV、3C（Priority SUMO enzyme digestion）.

A: Increase incubation time: Provide sufficient binding time between protein and filler; Increase packing volume: Increase the probability of collision between protein and packing, thereby improving binding efficiency.

A: Increase the concentration of salt ions in the buffer solution (<2M) during the purification process to aid dissolution; Add DTT (<100 mM) to open disulfide bonds and prevent protein aggregation and precipitation; Add protective agents such as glycerol or sucrose (5% -10%).

A: Plasmid quality: endotoxin level, presence or absence of protein or nucleic acid contamination, supercoiled ratio, etc.

Protein intrinsic factors: cytokines, capsid proteins, hormones, membrane proteins, or proteins with excessive or insufficient molecular weight.

Cell state, observe for contamination, cell density, etc.

A: You can try from the following aspects.

Try replacing different signal peptides.

Add large tags such as MBP/Fc to the N-terminus of the target sequence. MBP may not hang on the column in eukaryotic systems and requires fusion with other tags for purification, while Fc tags can be used for purification.

A：Liquid or freeze-dried.

Synbio Technologies offers e. coli, yeast, mammalian, and baculovirus/insect cell expression. Our team will work together with you to select the most suitable system for your protein and project.