Protein and Antibody

1. Sequencing results, including the sequence and chromatogram.
2. Plasmid vector name.
3. Recombinant cloning plasmid (10 µl) or expression plasmid (20 µl) with concentration >100 ng/µl.
4. Plasmid antibiotic resistance.
5. Primers used for cloning and sequencing.

(1) Obtaining the target gene.
(2) Plasmid construction.
(3) Strain screening.
(4) Expression condition optimization.
(5) Protein purification.

A vector is a self-replicating DNA molecule used in genetic engineering recombinant DNA technology to transfer a DNA fragment (target gene) into host cells. The three most commonly used vectors are bacterial plasmids, bacteriophages, and animal/plant viruses. The type of vector must match the host. Based on the host, they are classified as prokaryotic (bacterial) expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors, insect expression vectors, etc. The vector contains the foreign gene fragment. Through vector mediation, the foreign gene can be expressed in the host. GeneCreate primarily uses prokaryotic expression vectors.

You can choose liquid form or lyophilized powder, Protein lyophilization incurs an additional fee. Lyophilized powder is reconstituted with deionized sterile water; fully dissolve it. The specific concentration depends on your experimental requirements.

1.Post-translational modifications such as phosphorylation and glycosylation can increase molecular weight.
2.Post-translational cleavage by the protein itself can decrease molecular weight.
3.Some proteins have several splice variants, each with a different molecular weight; these should be distinguished.
4.Related to the amino acid composition (charged vs. non-charged).
5.Formation of multimers can increase molecular weight.

Fusion tags are used for detecting and purifying the target protein. Sometimes they are also used to increase the solubility of the target protein in the cytoplasm or to assist in transporting the target protein to the periplasm to enhance its biological activity. Common soluble tags like GST, MBP, and SUMO are often added to prokaryotic expression vectors or protein sequences. These tags are highly soluble when expressed in the host. When fused to a foreign protein, they can promote the soluble expression of the foreign protein.

Factors affecting prokaryotic expression include: translation initiation site, GC content, mRNA secondary structure, codon bias, choice of plasmid vector, gene/protein size, toxicity of the foreign gene to the host, gene mutations (frameshifts), and culture/induction conditions.

The main reason recombinant proteins often fail to fold correctly in the E. coli cytoplasm is the reducing environment, which disfavors disulfide bond formation. To address this, researchers have mutated genes of key enzymes in the main reducing pathways for intracellular expression. This has led to the construction of competent cells that favor disulfide bond formation and correct protein folding, such as Origami(DE3), Origami B(DE3), Rosetta-gami(DE3), Shuffle, etc.

When recombinant proteins are expressed at high levels in E. coli, they easily form insoluble inclusion bodies within the cells, causing trouble for subsequent purification and refolding. Moreover, the refolded protein often has low or no activity. Therefore, improving the soluble expression of recombinant proteins in E. coli is very necessary.
Optimization can be done from the following aspects:
1.Selecting an appropriate host cell.
2.Using suitable fusion tags.
3.Choosing an appropriate plasmid vector.
4.Optimizing culture and induction conditions.

Intracellular expression is the primary form of recombinant protein expression in E. coli. However, the E. coli cytoplasm has a reducing environment, which is unfavorable for the formation and stability of protein disulfide bonds. This leads to incorrect protein folding and the formation of insoluble inclusion bodies. Protein kinetic model studies indicate that the yield of active protein also depends on the rates of protein synthesis, folding, and aggregation. When foreign proteins are highly expressed in E. coli, if the aggregation rate of nascent polypeptide chains exceeds their folding rate, it leads to inclusion body formation.