Expression of recombinant proteins in E. coli is often the fastest and most cost-effective solution for recombinant protein production. KMD Bioscience leverages many years of experience in bacterial expression services to deliver the highest quality recombinant protein. In order to ensure the efficiency and activity of proteins, the following aspects are usually involved:

Question 1：Host system selection

When selecting a host system for expressing protein there are many factors to consider. These include: post translational modifications, equipment availability, and time constraints. We will choose different hosts according to client requirements, such as bacteria, yeast, filamentous fungi and unicellular algae. Each type of host has its own merits and drawbacks. For example, E. coli expression system is not suitable for post-translational modification (PTM) of proteins, but its advantages are as followed:

Fast propagation: The population of a bacteria culture doubles every 20 minutes and it takes only a few hours to reach the stationary phase.

E. coli cells easily achieve high cell density.

High efficiency of plasmid transfection.   

Question 2：Vector selection

At present, the most commonly used expression vectors incorporate a replicon, a promoter, a selection marker, a multiple cloning site (MCS) and a fusion tag removal strategy, as shown below:

Replicon: Contains the origin of replication and cis-acting control elements. When selecting a suitable plasmid vector, the copy number of a plasmid should be an important parameter that we need to consider. We have several vector series, such as pET, pUC, pACYC, pBAD and pSC101. The pET vector contains a pMB1 promoter, which usually has 15-60 copies in a single cell. The pUC vector contains a mutated pMB1 promoter, which typically has 500–700 copies in a single bacterium. PACYC and pBAD series are often used in dual expression systems, such as FRET system, which contains a p15A promoter and 10-12 copies in a single cell. The copy number of pSC101 vector in a single cell is usually < 5, and is commonly used for co-expression of three proteins. Combining the characteristics of these plasmids, our scientists obtained a bifunctional expression vector (Bi-plasmid vector) by engineering the pET vector, which contains dual MCS sites, T7 promoters, lac operons and ribosome binding sites. Using this plasmid, we can perform a transfection for the client to obtain independent expression of the two proteins.

Promoter: The lac promoter is one of the most commonly used promoters in E. coli expression system. We ofen use the pET vector with T7 promoter to express the target protein. When the plasmid containing T7 was transfected into bacteria cells, monoclones were selected and cultured, and IPTG was added to induce bacterial expression of recombinant protein.

Selection marker: Generally, the resistance genes used in the plasmid vector include ampicillin, kanamycin, chloramphenicol, gentamicin and tetracycline. During the expression and purification of recombinant proteins, most of the foreign antibiotics were naturally degraded or removed, leaving only a small amount of residue (<1 pg/ml) and we developed an ELISA kit could quickly detect antibiotic residues.

Affinity label: Affinity labels are used in the expression of recombinant proteins to make the protein purification easier and promote the solubility of certain insoluble proteins. Protein labels can be divided into two categories, one is short-peptide labels, the other is long. The long-peptide label could improve protein solubility, and it is often necessary to add short-peptide label to help protein purification. The short-peptide label generally contains only a few amino acids with molecular weight less than 2 kDa, which has little effect on the properties of recombinant protein. Short peptide tags generally contain only a few amino acids with molecular weights < 2 kDa, which have little effect on the properties of the recombinant protein. The affinity tag can be placed at the C-terminus and N-terminus of the protein. If a secreted protein is desired to be expressed, it is placed at the C-terminus and the signal peptide is placed at the N-terminus.

Remove labels: Protein labels may not be removed during general biological studies. For example, GST pull down and CO-IP experiments require retention of the GST and His tags to separate the target protein directly from the mixture. But when it comes to protein structure studies, protein labels or fusion partners need to be removed. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing.  TEV protease with His label has gradually been widely used in label removal experiments. The protease has an excellent feature of leaving only one Ser or Gly residue or no residual amino acid residue after excision. This is also a commonly used protein label removal method in KMD Bioscience.

Question 3：Choosing the right host for protein expression

Prokaryotic expression of recombinant protein often uses BL21 (DE3) and K-12 derived strains as expression hosts. BL21 (DE3) has been the gold standard for protein expression since it was first introduced in 1990. Deficient in lon and ompT proteases, BL21 and its derivatives are ideal for many applications. K-12 strains are by far the most frequently used host strains in gene cloning experiments, since they have the following advantages: (i) they represent the genetically best understood living organism, (ii) they are easily modified by many genetic methods, and (iii) they are classified as biologically safe vehicles for the propagation of many efficient gene cloning and expression vectors in all major national and international guidelines on biological safety for work with recombinant DNA technology.

Question 4：Strategies for successful expression of a recombinant protein

The expression of recombinant proteins is often not as smooth as theory, and various problems may arise in the actual expression process. When it is very difficult to choose an optimal strategy, using a seemingly clumsy method may become an unexpected shortcut to success. For example, if two recombinant proteins are required to be expressed, then constructing six different expression plasmids means that there are 36 different expression conditions. The best combination of conditions can be obtained by high-throughput microprotein expression assay at one time, then subsequent experiments and cost control will become easier.

Service Highlights:

*Codon optimization: including rare codon analysis, protein hydrophilicity analysis and prediction of transmembrane domains.

*A variety of fusion tag vectors and hosts to ensure high soluble protein expression and activity.

*Scale-up: from 500ml to 500L.

*Short cycle and high output.

*LAL <0.1EU/mg.

*Customization: quantity, purity, tags, and endotoxin levels are customizable and RNase removal is optional.

References: Rui Zhai, Xiaoyun Gong, Jie Xie, et al. Ultrasensitive analysis of heat shock protein 90α with antibodies orderly arrayed on a novel type of immunoprobe based on magnetic COFs.[J]. Talanta, 2019, 1(191):553-560.