1. What is recombinant protein expression?

It reveals that life activities can be explored from different levels such as genes and proteins. With the continuous development of science and technology, protein research has developed from protein extraction to recombinant protein expression. At present, we can use a variety of recombinant protein expression systems (prokaryotic expression system, yeast expression system, insect expression system, mammalian expression system, plant expression system) to recombinant expression, separation and purification of our target proteins to further explore the function of proteins, providing a convenient method for scientific research (Belenkaya S. V, 2023).

2. Experimental procedure of recombinant protein expression

The main components include expression vectors and host cells, and the main experimental procedures are as follows:

(1) Construction of expression vector.

The selection of expression vector is usually based on the purpose of the expressed protein, known information and cloning and purification strategies. After the vector is selected, the target gene is connected to the expression vector by homologous recombination or seamless cloning.

(2) Transform host cells.

Host cell selection can be based on the characteristics of the host strain as well as the use of the target gene expression product. For example, when there are more proteases produced in the host cell, which affects the stability of the target gene expression product, the protease-deficient strain can be selected, such as BL21 series is suitable for the expression of toxic proteins. When the foreign gene to be expressed is a eukaryotic gene, which is difficult to be expressed in prokaryotes, the Rosetta series strains supplemented with rare codons can be selected. After selecting a suitable host strain, the constructed plasmid containing the target gene is transformed into the host cell, and the positive clone is screened.

(3) Induced expression of target protein

Culture conditions are the key factors for successful production of recombinant proteins. IPTG is the commonly used induction method for prokaryotic expression, and methanol is the commonly used induction method for yeast expression. However, exploration and optimization should be carried out for different target genes and host strains, the concentration of inducers, and the culture temperature and time to achieve the best culture conditions.

(4) Separation and purification of target protein.

According to the properties of proteins, different expression forms and fusion labels, suitable methods were selected to isolate and purify the target proteins.

(5) Detection and identification of target protein

The size and purity of purified protein can be detected by SDS-PAGE and Western-blolt methods. All procedures should be performed on ice to prevent protein denaturation.

KMD Bioscience has been committed to the expression, separation and purification of recombinant proteins for many years, and has built protein platforms such as prokaryotic expression system, yeast expression system, insect expression system, mammalian expression system and plant expression system for different protein expression needs. For protein expression purification, we can provide the following services and protein expression solutions.

This article serves as a reference material for enthusiasts in scientific research. It does not substitute for professional knowledge or hands on experimental procedures which require more detailed and professional information. In case of any content infringement, kindly reach out to the author for immediate deletion of the contentious material.

References:

Belenkaya S. V., et al.”Comparison of the Biochemical Properties of Recombinant Alpaca (Vicugna pacos) Chymosins Produced in Pro- and Eukaryotic Expression Systems.”Applied Biochemistry and Microbiology 59.5(2023):630-635.

Suo Qian, et al.”[Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae)]..” Sheng wu gong cheng xue bao = Chinese journal of biotechnology39.7(2023):2730-2742.

Wu Yijian, et al.”An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.”Frontiers in Bioengineering and Biotechnology 11.(2023):1249841-1249841.