Recombinant Protein Production Protocol and FAQs

Recombinant Protein Production is a process of expressing foreign genes and producing target proteins in host cells by genetic engineering. This technology is usually used to prepare proteins that are difficult to obtain or cannot be effectively produced in the natural state, such as various proteins for medical, scientific, industrial and biotechnology applications.

The Principle of Recombinant Protein Production

Based on genetic engineering technology, exogenous genes are expressed and target proteins are produced by using the transcription and translation mechanisms of host cells. The core process is to insert a gene encoding a specific protein (usually a foreign gene) into the host cell and produce the target protein through the natural biosynthesis process of the cell.

The Steps of Recombinant Protein Production

The experimental steps of recombinant protein production usually include the following key steps. The successful completion of each step is crucial to the final high-quality recombinant protein:

Gene Cloning and Vector Construction

Selection and extraction of target genes

The coding genes of target proteins were extracted from source organisms (such as humans, bacteria, plants, etc.). The target gene is often amplified by PCR (polymerase chain reaction).

Insertion of an expression vector

The target gene is cloned into an expression vector (such as a plasmid). Expression vectors usually contain promoters, tag sequences, selectable markers, etc., which can drive the expression of foreign genes in host cells.

Vector verification

The constructed vector was digested and sequenced to ensure that the target gene was correctly inserted into the vector.

Selecting the Appropriate Host Cells

According to the nature of the target protein and production needs, select the appropriate host cells. Common hosts include:

1 Escherichia coli (E.coli) : suitable for simple protein production and low production costs.
2 Yeast, insect cells, mammalian cells: suitable for the production of complex proteins, such as proteins requiring glycosylation, folding and complex modification.

Transformation/Transfection of Host Cells

Select the appropriate host cells

Select the host cells according to the characteristics of the target protein. Commonly used host cells include Escherichia coli (E.coli), yeast, insect cells, mammalian cells, etc.

Prokaryotic transformation (for prokaryotic hosts such as E.coli)

Expression vectors are introduced into host cells by heat shock, chemical transformation, or electroporation.

Transfection (for eukaryotic hosts, such as mammalian cells)

The vector is introduced into host cells using chemical reagents (such as liposomes) or electroporation techniques.

Screening of transformed/transfected cells

Successfully transformed or transfected cells were selected by antibiotic screening (such as using antibiotic resistance genes contained in the vector).

Protein Expression

Induced expression

If the host cell used has a controllable expression system (such as the T7 system in E.coli), the expression of the target protein is activated by adding inducers (such as IPTG).

Optimization of expression conditions

The expression level and folding state of the target protein were optimized by adjusting the culture temperature, medium composition, induction time and other conditions.

Expression check

SDS-PAGE, Western blot and other methods were used to preliminarily check the expression of the target protein to ensure its expression quantity and quality.

Cell Collection and Lysis

Cell collection

The host cells are separated from the culture medium by centrifugation and usually frozen or directly used for lysis.

Cell lysis

The cells were disrupted by physical methods (such as ultrasonic disruption, high pressure homogenization) or chemical methods (such as lysis buffer, lyase, etc.) to release the contained proteins.

Check the lysis effect

The lysis solution was analyzed by SDS-PAGE to confirm the release of the protein.

Protein Purification

Preliminary purification

Select the appropriate purification method according to the characteristics of the target protein. Commonly used technologies include:

1 Affinity Chromatography : Selective purification of the target protein by binding the affinity tag (such as His tag) to the affinity column.
2 Ion Exchange Chromatography : Different proteins are separated according to the charge characteristics of the protein.
3 Gel Filtration Chromatography : Separation based on the molecular weight of the protein.

Verification after purification

SDS-PAGE, Western blotting and other techniques were used to confirm the purity and molecular weight of the target protein.

Protein Activity Verification and Characterization

Functional verification

Biological methods were used to verify whether the protein has the expected biological activity. For example, enzyme activity assay, antibody binding assay, etc.

Structural characterization

The structure of the protein was further analyzed by mass spectrometry, nuclear magnetic resonance (NMR) or X-ray crystallography.

Protein stability evaluation

The stability of the protein was evaluated by thermal stability, pH stability and other experiments.

Protein Storage and Application

Storage conditions

According to the characteristics of the protein, select the appropriate storage conditions (such as cryopreservation, freeze-drying, etc.) to ensure the stability of the protein.

Application

The purified recombinant protein can be used for various applications, such as scientific research, vaccine development, enzymatic applications, drug production, etc.

Figure 1. Basic expression vectors for high-throughput expression in E. coli of (a) cytoplasmic proteins and (b) membrane proteins. (Reference source: High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives.）

Alpha Lifetech provides Recombinant Protein Production services, providing customers with high-quality recombinant protein production solutions, covering the entire process from gene cloning, expression system selection to protein purification and quality control. We are committed to providing customized recombinant protein services for biopharmaceuticals, scientific research institutions and pharmaceutical companies to meet the needs of customers at different research stages.

FAQ

1. Low expression of recombinant protein.

Cause: protein expression system (such as Escherichia coli, yeast, insect cells, etc.) improper selection. Improper regulation of exogenous gene expression (e.g. inappropriate promoter selection). The target protein may be toxic and inhibit the growth of host cells. Mutations or post-translational modifications of the gene sequence affect expression. Solution : Choosing a more suitable expression system, such as insect cell systems, is usually better for expressing complex eukaryotic proteins. Use stronger or more suitable promoters to optimize the coding genes. Low temperature culture (such as 16℃) was used to reduce toxicity and improve protein folding. Mutation analysis or tag optimization of genes.
2. Protein failed to fold correctly, resulting in the formation of inclusion bodies.

Cause: protein overexpression, resulting in folding mechanism can not keep up. The expression system does not support the correct folding of the target protein. The protein structure is complex and requires auxiliary molecular chaperones to help fold. Solution : Lower expression temperature (e.g. 16℃ or lower) slows down the rate of protein synthesis and increases the probability of correct folding. Co-expression of molecular chaperones (such as GroEL / GroES) or folding factors helps proteins fold correctly. Choose the appropriate expression system, such as expression in insect cells or mammalian cells. Chemical modifiers or medium additives were used to promote folding.
3. Difficulties in protein purification.

Cause: The target protein is insoluble or poorly hydrophilic, making it difficult to extract. Proteins have similar physical and chemical properties to host proteins and are difficult to separate. The loss is serious in the purification process. Solution : Improve the specificity and efficiency of purification by tag purification methods (such as His tag, GST tag). The buffer and salt concentration were optimized to ensure the stability of the protein in the solution. A combination of affinity chromatography, ion exchange chromatography, gel filtration and other purification methods was used.
4. Protein degradation or inactivation.

Cause: The target protein is degraded by enzymes inside the cell or during the purification process. Proteins are unstable in solution. Solution: Add protease inhibitors to prevent degradation. Adjust the temperature, pH, and ionic strength during the purification process to avoid protein degradation. Optimize the stability of the target protein (such as by fusing tags or adding stabilizers)- Store proteins at 80 ℃ to prevent denaturation.
5. Protein has no biological activity.

Cause: protein folding is not complete, unable to form active structure. Lack of necessary post-translational modifications (such as phosphorylation, glycosylation). The target protein requires a dimer or multimer structure to be active. Solution : Optimize the protein folding conditions to ensure that it has an active conformation. Post-translational modifications are performed in appropriate expression systems, such as expression in mammalian cells to ensure glycosylation. To examine and regulate the conformation of a protein, using appropriate cofactors or chemical modifications.

reference

[1] Ferrer-Miralles N, Saccardo P, Corchero JL, Xu Z, García-Fruitós E. General introduction: recombinant protein production and purification of insoluble proteins. Methods Mol Biol. 2015;1258:1-24. doi:10.1007/978-1-4939-2205-5_1

[2] Kosobokova EN, Skrypnik KA, Kosorukov VS. Overview of Fusion Tags for Recombinant Proteins. Biochemistry (Mosc). 2016;81(3):187-200. doi:10.1134/S0006297916030019

[3] Hayat SMG, Farahani N, Golichenari B, Sahebkar A. Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know. Curr Pharm Des. 2018;24(6):718-725. doi:10.2174/1381612824666180131121940

[4] Jia B, Jeon CO. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol. 2016;6(8):160196. doi:10.1098/rsob.160196

[5] Jenkins N, Meleady P, Tyther R, Murphy L. Strategies for analysing and improving the expression and quality of recombinant proteins made in mammalian cells. Biotechnol Appl Biochem. 2009;53(Pt 2):73-83. Published 2009 May 6. doi:10.1042/BA20080258

[6] Wingfield PT. Overview of the purification of recombinant proteins. Curr Protoc Protein Sci. 2015;80:6.1.1-6.1.35. Published 2015 Apr 1. doi:10.1002/0471140864.ps0601s80

[7] Oliveira C, Domingues L. Guidelines to reach high-quality purified recombinant proteins. Appl Microbiol Biotechnol. 2018;102(1):81-92. doi:10.1007/s00253-017-8623-8