Protein Expression and Purification Services

The field of protein expression and purification is pivotal in advancing biological science research and is gaining significant traction in foundational studies, drug development, and the biotechnology sector. Our team boasts profound expertise and cutting-edge techniques to ensure the efficient and high-quality expression and purification of proteins.

Creative BioMart provides you all kinds of expression systems that can produce your recombinant proteins including monoclonal antibodies, vaccine antigens or your disease biomarkers, and even produce your novel proteins. All kinds of expression systems are adopted for different purposes according different requirements including structural studies, functional assays, target validation, high throughput screens.

What is Protein Expression and Purification?

Protein expression and purification service refers to a biotechnology service based on bioengineering technology to make the vertex target protein have the effect of high expression and purification, and obtain high quality protein sample for the purpose of scientific experiments and industrial purpose. Expression and purification are mainly the process of expressing the identified target protein by the host cell culturing in-vitro, such transplanted gene can make the cell express the identified protein, and then a series of purification step will make the protein become out of impurities such as cell membrane and other non-target proteins, to obtain the high purity target protein.

Purified proteins play a vital role in industrial development and scientific research. First, as the core executor of cell functions, studying the structure and function of proteins can help scientists reveal various physiological and pathological phenomena in life activities. In addition, purified proteins have a wide range of commercial applications, including drug development, enzyme engineering, and vaccine production. In addition, high-quality protein samples are indispensable key materials for structural biology and functional research.

In offering protein expression and purification services, it is essential to pay particular attention to certain standards and considerations. Selecting an appropriate expression system-be it E. coli , yeast, insect cells, or mammalian cells-is vital, as is devising a purification strategy. Decisions on these fronts should be informed by the properties of the target protein, its intended applications, and the associated economic implications. There are also several important service quality evaluation indicators, including expression level, purification yield, protein activity, and stability. Using a suitable buffer system during purification to prevent protein degradation and inactivation is a key step to ensure success.

Common Protein Expression Systems

Bacterial System

Bacterial expression system, especially E. Coli , is one of the most commonly used protein expression systems, which takes advantage of the rapid growth of bacteria and high genetic engineering operability.

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Yeast System

The yeast expression system combines the advantages of bacterial and mammalian systems and is simple and easy to use, making it a reliable choice for protein expression.

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Insect System

The insect expression system uses insect cell culture to express complex eukaryotic proteins and is suitable for the production of a variety of proteins.

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Mammalian System

Mammalian expression systems provide the protein expression pathway that is closest to the native state and are suitable for the preparation of proteins with high value and high functional requirements.

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Cell-free System

The cell-free protein expression system synthesizes proteins directly from template DNA/RNA in an in vitro environment without relying on living cells. It is a fast and flexible expression method.

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Protein Purification Techniques

Protein purification strategies are essential for separating and characterizing proteins from intricate biological samples, which facilitates a deeper understanding of their structure, function, and how they interact with other molecules. The process often begins with the concentration of proteins from raw extracts through initial procedures such as centrifugation and precipitation. Following this, proteins are further refined through chromatographic techniques, which sort proteins according to their specific attributes. The principal types of chromatography used in this context include:

Ion exchange chromatography is a widely used technique in protein chromatographic analysis. It is based on the charge difference of the protein to achieve separation. When proteins pass through a column of charged resin, those with the opposite charge of the resin are attached to the resin, while other proteins flow directly through it. By carefully adjusting the salt concentration or pH, those adsorbent proteins can be selectively released, enabling a precisely controlled elution process.

High Performance Liquid Chromatography (HPLC) is highly regarded for its superior accuracy and high efficiency. For protein purification by HPLC, the protein mixture is injected into a column containing a stationary phase under high pressure. Proteins are separated based on their different interactions with stationary phases, which may involve molecular size, charge, hydrophobicity, or specific affinity.

Gel filtration chromatography also known as size exclusion chromatography, is a technique for separating proteins based on their size and shape. This method uses porous particles of different pore sizes in a gel filter column. When a mixed protein passes through the filter column, smaller molecules can enter the pores of the particles and are delayed in flowing out, while larger molecules cannot enter these pores and flow out of the filter column faster. This technique is particularly suitable for removing aggregates or degradation products from samples, or for determining molecular weight.

Affinity chromatography employs a separation strategy that capitalizes on the specific biological interactions. It leverages the highly selective affinity between proteins and their corresponding ligands to facilitate the separation, like the interaction between enzymes and their substrates, antibodies and antigens, or receptors and their ligands. In the setup of the affinity chromatography column, these particular ligands are permanently bonded to an inert support matrix. When a mixture containing the target protein flows through the affinity column, the target protein will bind specifically to the ligand, while other proteins will be eluted. The bound protein can then be released by adjusting the elution conditions, such as changing the pH or ionic strength.

Instrument Platform

Creative BioMart is equipped with a series of biochemical instruments and various tools to assist in protein expression and purification services. Centrifuges and ultrasonic disruptors are used to separate bacteria/cells and release proteins, chromatographic systems are used for protein separation and purification, ultrafiltration equipment is used for concentration and desalting, and spectrophotometers and mass spectrometers are used to detect protein purity and molecular weight, etc. In addition, there are CO ₂ incubators, PCR machines, electrophoresis equipment, WB detection systems, etc. To ensure satisfactory service results, we will also strictly control operating conditions, monitor key steps, and regularly calibrate and maintain instruments.

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  Ultraspeed Refrigerated Centrifuge
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  HPLC System
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  PCR System
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  Ultrasonic Crusher
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  Imaging System

What We Offer?

We are committed to providing services at all levels, from upstream gene synthesis and cloning, categorical expression system, to midstream protein purification, and then downstream protein analysis , covering the whole process of protein research to ensure that the diversified needs of customers are met efficiently and accurately.

Popular Custom Protein Expression Services

Classification	Expression System
Common Expression Systems or Platforms	Bacterial expression Systems ( E. coli / Bacillus ) Yeast expression Systems ( P pastoris / S cerevisiae ) Baculovirus-Insect Cell expression Systems (SF9, SF21, S2, T.ni / HI-5)</a > Mammalian expression Systems (CHO / HEK293)		Cell-Free in Vitro Protein expression Serum-Free expression System Nicotiana tabacum Specific expression Platform Rice Endosperm Specific expression Platform
Bulk Protein Production	High-throughput Protein Production Large-Scale Protein Production Service	Mass Standard Protein Production High Yield Protein Production
Tagged Protein Production	Fc Fusion Protein Production	SUMO-Tag Protein Production
Special Functional Protein Production	Human Synthetic Proteins Produced in Mammalian Cell Lines</a > Pichia Glyco Protein Production	Albumin Fusion Protein Production Services
Vital Protein Production	Recombinant Antibody Production Services	Membrane Proteins Expression and Purification
Other Protein Production	Endotoxin-Free Protein Production	Fucose-Free Protein expression System

Purification Method

Depending on the requirements and nature of the protein, we are able to design specialized approach for extraction. We will purify the protein cost-efficiently, characterize it according to your specifications and deliver it to you in an active and application-compatible form. Purification protocols specified by our clients or obtained from the literature can also be followed at our site. Click here to find more about our Protein Extraction Services.

Types of Extraction	Extraction Modes	Protocols	We Deliver
Organelle protein extraction Nuclear protein extraction Membrane protein extraction Cytoplasmic protein extraction	Ion-exchange Gel filtration Affinity (broad-spectrum) Hydrophobic interaction	Client-supplied protocols Published protocols Improved protocols developed by Creative BioMart Protocols tailored to the client's requirements	Purified protein sample Specified protein concentration Special formulation to prevent protein from degradation Custom aliquoting as specified by customer Certificate of analysis as specified by customer

Protein Modification Services

In protein expression and purification services, protein modification is a key link, which can comprehensively improve the function and stability of proteins. The modification process can be carried out according to different needs, including but not limited to covalent modifications such as glycosylation , phosphorylation , methylation, ubiquitinylation and acetylation . These modifications usually occur after translation and can significantly affect the activity and stability of the protein as well as its intracellular localization. In addition, redox modification of proteins (such as disulfide bond formation) and protein engineering (such as point mutations) are also common methods used to improve protein solubility, thermal stability, and functional activity. Among the services we provide, we also include labeling through specific chemical modifications for subsequent tracking and detection, which can greatly facilitate downstream applications, such as structural biology research, drug development, and protein-protein interaction analysis. Our professional technical team can also customize different modification plans according to the specific needs of customers to ensure the effectiveness and pertinence of the modification.

Protein Analytical Services

Classification	Research Project	Method (Partial)
Protein Characterization	Protein Identity, Purity, Impurities	SDS electrophoresis, Analytical Size-Exclusion Chromatography, HPLC
	Charge Isoform	Gel Isoelectric Focusing, Capillary Isoelectric Focusing, OFFGEL electrophoresis
	Aggregation analysis	SDS PAGE, Chip-based Protein Electrphoresis
	Oxidation	HPLC-LC/UV, Reversed-Phase LC/MS
	Amino Acid Analysis	OPA derivitization + RP-LC/UV, FMOC derivitization + RP-LC/UV
	Peptide Mapping	RP-LC/UV, RP-LC/MS
	Glycan Profiling	HPLC/fluorescence detection, MALDI-TOF/MS, LC/MS
Protein Interaction	Standard Techniques	Co-IPs, GST-pull downs, Yeast 2-Hybrid
	In vivo lmaging	FRET, BRET
	Biophysical Techniques	MS, Surface plasmon resonance, NMR, X-ray crystallography
	High Throughput Techniques	Library screening, Degenerate peptide Libraries, Spot blots, Co-IPs, GST-pull downs, Yeast 2-Hybrid
Modification Assay	Protein Methylation	MS, ChIP, Methylation-Specific Antibodies
	Protein Acetylation	WB, MS, IP, ELISA
	Protein SUMOylation	WB, MS, IP, Fluorescent labeling, Pull downs, Yeast 2-Hybrid
	Protein Deubiquitination	WB, MS, IP, Pull downs
	Protein Phosphorylation	WB, Radiolabeling, MS, ELISA
Protein Activity	Protein Kinase	WB, ELISA, MS, HPLC, Fluorescence polarization assay
	ATPase/GTPase	MS, HPLC, Radioactive Assays, Colorimetric Assays, Fluorescent Assays

Service Procedure

Details for Service Procedure

1 Gene cloning and vector construction (1-2 weeks)

First, the target gene fragment is isolated from a specific resource and inserted into an expression vector, which usually requires the design of specific primers and the selection of the best vector. This step includes primer design, PCR amplification, vector selection, connection and transformation. The recombinant construct is confirmed by DNA sequencing. Codons can be optimized according to the expression system if necessary.

2 Expression system selection and optimization (1 week)

Choose the most appropriate expression system (bacteria, yeast, insect cells, mammalian cells, cell-free systems) to achieve optimal expression. Considerations include protein complexity, modification requirements, and yield.

3 Small-scale protein expression (1-2 weeks)

Use a small amount (like 5 mL) of cultured expression host to make the cloned plasmid express the target protein. And use SDS-PAGE or other protein detection methods to capture the target protein in the supernatant or precipitate to evaluate the gene expression level, protein expression level, protein secretion and protein solubility. We will deliver the protein analysis results of this link to you. If you are not satisfied, you can terminate the service at any time.

4 Large-scale protein expression and purification (2-4 week)

Scale up the culture volume to 2 L, collect the expressed bacteria/cells (or culture supernatant) and break them to release the protein. Use different purification methods (such as affinity chromatography, ion exchange, exclusion chromatography, etc.) to separate and purify the target protein to ensure high purity and activity. Also, we will verify and characterize the purified protein by SDS-PAGE, Western blot, mass spectrometry and other techniques.

5 Scale-up and process optimization (laboratory to industrial scale) (weeks to months)

After successfully obtaining purified protein, the process is scaled up to the required scale and production parameters are optimized to increase yield and reduce costs. The purified protein is prepared into a final formulation that can be used for research or industrial production, including protein concentration determination, buffer exchange and long-term storage solution optimization. The specific time consumption depends on the production volume, formulation requirements and storage conditions.

Case Study

SDS-PAGE Analysis of Target Protein

• Case 1: Recombinant Human GFAP aa. (Glu254~Glu374 (Accession# P14136)) fused with N-terminal His & S tag expressed in E. coli cells

Project: Tagged truncated protein
Requirements: Endotoxin <1.0EU per 1µg, purity >95% by SDS-PAGE
Service: Bacterial expression Systems ( E. coli / Bacillus )

SDS-PAGE and Activity Analysis of Target Protein

• Case 2: Recombinant Full length Human peptidylprolyl isomerase A (cyclophilin A) Protein, His-tagged expressed in HEK293

Project: Full length protein with His tag
Requirements: Endotoxin <1.0EU per 1µg, purity >95% by SDS-PAGE
Service: Mammalian expression Systems (CHO / HEK293)

SDS-PAGE and Activity Analysis of Target Protein

• Case 3: Recombinant Human ARAF (YY301, 302DD) Mutation Protein, GST/His-tagged expressed in Sf9 insect cells

Project: Recombinant protein containing mutations
Process: A GST-tag at the N-terminal end as well as a C-terminal His-tag (6xhis) and affinity purified
Specification: Bio-activity 6 pmol/min/μg and purity 61%
Service: Baculovirus-Insect Cell expression Systems (SF9, SF21, S2, T.ni / HI-5)

SDS-PAGE and Activity Analysis of Target Protein

• Case 4: Recombinant Human CCR4 Full Length Transmembrane protein (1-360 aa), His-tagged(VLPs) expressed in HEK293

Project: Full length transmembrane protein
Bio-activity: Measured by its binding ability in a functional ELISA. Immobilized Human CCR4 at 10 μg/mL can bind Anti-CCR4 recombinant antibody, the EC50 is 362.3-630.8 ng/ML
Service: Mammalian expression Systems (CHO / HEK293)

Why Choose Us?

Proprietary Technologies. The unique methods and patented technologies we use can improve the efficiency of protein expression, the accuracy of purification, and the stability and functional integrity of the final product.

Guarantees your protein expression and purification. We ensure production meets required protein yield and quality, provide transparent reporting of project progress, and provide rapid response and resolution mechanisms when problems arise.

Competitive price. While ensuring high quality, we also offer the most competitive prices in the industry.

Fast turnaround: as little as 5 weeks. It is estimated that the fastest service process will take 5 weeks. If there are other requirements, the time will fluctuate based on this.

Flexible scale-up protein production. We offer a variety of production options, from small-batch laboratory production to large-scale industrial production.

Customizable protein. We can modify the protein structure, function or expression system according to your special needs.

FAQs

Below are some frequently asked questions about our Protein Expression and Purification Services. If you have any other questions, please contact us and we will be happy to assist you.

• Q: How long does it usually take to produce custom proteins?

  A: We usually complete custom protein production within 6 to 8 weeks. However, this timeframe can change based on the complexity of the protein's characteristics and whether extra services like endotoxin removal are needed.

• Q: What quality control information do you offer and how is the purity measured?

  A: We assess the quality and determine the purity of custom proteins using SDS-PAGE and Coomassie staining. Proteins with His-tags are additionally evaluated through western-blot analysis, whereas tag-free proteins may be subject to optional LC-MS/MS analysis upon request.

• Q: In addition to the His tag, what other tags do you offer for expressing recombinant proteins?

  A: We provide commonly used tag proteins including His-tag, GST-tag, MBP-tag, FLAG-tag, Myc-tag, HA-tag, etc. to promote protein purification, solubility and detection.

• Q: What if I need the tag removed?

  A: If the tag needs to be removed, we offer extensive production and purification services, including tag removal and endotoxin removal.

• Q: Where does the expression of a protein begin?

  A: Creative BioMart can initiate the process with gene synthesis for protein expression, requiring either the target protein's sequence data or its Uniprot/NCBI reference number. We can also offer gene optimization services upon your request. If you possess the plasmid, we can utilize the provided template DNA or the ready-to-express construct for your project.

Resources

Creative BioMart provides customers with free resources related to recombinant protein expression. Get additional information to help you advance your studies here.