Strategies for Construction of Recombinant Escherichia coli Expression Vectors and Optimization of Cultivation Conditions

Preface

Protein expression in E. coli

(Source: Reduced background expression and improved plasmid stability with pET vectors in BL21(DE3). BioTechniques 29, 1234-1238)

In this post-genomic era, protein expression and purification play a pivotal role in biochemistry. Recombinant proteins can be expressed through prokaryotic systems (Escherichia coli and Bacillus subtilis), eukaryotic systems (yeast, insect cells, and mammalian cells), or in vitro systems. The E. coli system is the preferred host for initial screening of recombinant protein expression due to its ease of manipulation, low culturing costs, and rapid growth. In recent years, numerous novel strains, vectors, and tags have been developed to overcome the limitations of this system, including codon bias, inclusion body formation, toxicity, protein inactivation, mRNA instability, and the lack of post-translational modifications. In this article, we will focus on the strategies for constructing expression vectors and optimizing cultivation conditions in the E. coli expression system.

Construction of Expression Vectors

A complete expression vector mainly consists of basic components such as an origin of replication, a selective screening marker, a promoter, an inserted target gene fragment, and a transcription terminator. An expression vector with good performance generally has the following characteristics: high copy number, wide range of applications, and good stability in host cells.

Acquisition of Target Genes:

1. Obtained from a cDNA library after reverse transcription

2. Acquired from genomic DNA through PCR

3. Using array-based oligonucleotide assembly technology to synthesize custom genes

One of the main advantages of gene synthesis is that researchers can freely design genes of interest without the limitation of using natural templates. Additionally, using codon-optimized genes can ensure reliable expression, resulting in increased protein yield and solubility.

Selection of Promoters:

Effective promoters for expressing foreign proteins in E. coli possess four key characteristics:

1. Sufficient promoter strength

2. Minimal basal transcription activity

3. Simple induction methods

4. Precise regulation of activity

The T7 promoter in E. coli exhibits strong activity, allowing recombinant proteins to accumulate up to 50% of the total cellular protein. The pET expression system is currently the most widely used heterologous expression system.

5'UTR and N-terminal Codon:

Protein expression is initiated through the binding of ribosomes to the Shine Dalgarno (SD) sequence located in the 5'UTR. The spacing between the SD sequence and the start codon has a significant impact on translation efficiency and protein yield.

Selection of Fusion Tags:

1. Easy detection of protein expression

2. High protein expression and solubility

3. It is easy to isolate high-purity proteins from E. coli. A wide range of tags has been developed, and the general characteristics of commonly used tags are listed in the following table.

E. coli Host Strains:

Selecting an appropriate host plays a crucial role in protein expression, solubility, and yield. To date, various E. coli strains have been engineered to significantly enhance the production of membrane proteins. The following table summarizes commonly used E. coli hosts and their characteristics.

Culturing of E. coli

Optimization Strategies for E. coli Cultivation Processes:

1. Composition of the culture medium

2. Optimization of culturing conditions

3. Control of metabolic products

Main Components of the Culture Medium:

Carbon source, nitrogen source, water, inorganic salts, and trace elements. Different concentrations and ratios of these components can significantly impact the growth of recombinant strains and the expression of foreign genes. Among them, the ratio of carbon source to nitrogen source is extremely important. Controlling the carbon/nitrogen ratio can regulate bacterial growth and protein expression, improving conversion rates and reducing costs. Commonly used E. coli culture media include LB, TB, SB, MBL, etc. Typically, the inhibitory concentrations of carbon and nitrogen sources as well as various metal ions are as follows: Glucose 50 g/L, NH3 3g/L, Mg²+8.7g/L、PO3 4-10g/L、Zn²+0.038g/L、Fe²+1.15g/L.

Culturing Conditions:

PH, temperature, dissolved oxygen, and feeding rate are all critical culturing conditions. Both pH and temperature can affect the activity of various enzymes within the bacterial cells, thereby influencing the synthesis of metabolites and the target protein. The level of dissolved oxygen is also an important factor affecting the conversion rate, as both excessively high and low levels are detrimental to fermentation. The feeding rate is closely related to the conversion rate, and an appropriate feeding rate is key to improving the conversion rate and reducing production costs.

Control of Metabolites:

Acetic acid is a metabolic byproduct during E. coli fermentation, and concentrations as low as 5-10g/L can have observable inhibitory effects on lag phase, maximum specific growth rate, bacterial concentration, and final protein yield. The higher the specific growth rate, the more acetic acid is produced. Methods such as lowering the temperature, adjusting the pH, and controlling the feeding rate can be employed to reduce the specific growth rate, thereby inhibiting the production of acetic acid. Glucose is one of the essential carbon sources during E. coli fermentation, and maintaining it at a lower level can reduce the production of acetic acid.

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Protein Expression and Purification

Basic Process of Protein Expression (in Escherichia coli) and Purification:

Purification Experimental Results (Taking Collagen Project as an Example):

In addition, the E. coli expression projects also include the expression and purification studies of Protein A, Protein L, GLP-1, SNAP25 recombinant protein, and GFP.

Service

The project types include: antibodies (monoclonal/bispecific/trispecific), vaccines (VLP vaccines, subunit vaccines, etc.), blood products, recombinant proteins (collagen/others), etc. BioLink is capable of integrating upstream construction with downstream separation and purification to complete the development and optimization services for the entire product production process.