Proteins play fundamental role to life and they control all biochemical reactions and provide structure to organisms, in addition, proteins act as transport vital molecules and even defend the organism as antibodies. Depend on the central dogma of molecular biology, the blueprints for proteins are stored in DNA and then an intermediary template, messenger RNA (mRNA), is required to transfer the genetic information from the DNA to the protein via the transcriptional and translational process.
• Strain selection
• Codon optimization
• Fusion systems
• Isotope labeling
• Soluble Protein
• Folding of insoluble protein
• Membrane proteins
• Cleavage of fusion moieties
Protein Expression: The first step, protein expression aims at a large amount of proteins. This gene-to-protein process contains two main steps: Transcription and Translation. See Protein Expression. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. In order to get enough amount of the protein of interest, strain selection, codon optimization, fusion systems, co-expression, mutagenesis and isotope labeling techniques are usually used.
Protein Purification: The next step, protein purification, is to isolate the protein from the samples such as cell lysates or medium. Protein refolding, cleavage of fusion moieties and chromatography techniques are involved in protein purification process.
Protein Purification is focused on chromatographic purification of proteins at research scale. Before a specific protein can be identified and its properties can be studied, the protein must usually be separated and purified, where series of processes intended to isolate one or a few proteins from a complex mixture. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. The purification scheme of a protein must be optimized to complete this process in the least number of steps.
Protein yield and activity can be maximized by selecting the right lysis reagents and appropriate purification resin. Most recombinant proteins are expressed as fusion proteins with short affinity tags, such as polyhistidine or glutathione S-transferase, which allow for selective purification of the protein of interest.
Protein Production is a complex system of biotechnologies that each step influences with each other process. The recombinant protein purification method are mostly depend on the characteristics of the recombinant protein and the expression system applied. Sino Biological offers one-stop service from gene to purified protein with over 10 years' experience of protein expression and multiple protein purification and refolding technologies. We have got multiple protein expression systems such as bacterial, yeast, baculovirus-insect and mammalian expression system and 30+ purification systems to handle from 2 to 1000 L to meet high-throughput and large-scale protein expression and production.
Protein production is the biotechnological process of generating a specific protein. Commonly used protein production systems include those derived from bacteria, baculovirus/insect cells, mammalian cells and yeast.
Using the right expression system for your specific application is the key to success. Protein solubility, functionality, purification speed, and yield are often crucial factors to consider when choosing an expression system. Additionally, each system has its own strengths and challenges, which are important when choosing an expression system.
Protein purification means protein fractionation which is also called downstream processing. Protein purification involves a number of processes, including pumping and ultrafiltration, which involve significant shear environments. More importantly, protein tags are a useful and convenient tool for improving solubility of recombinant proteins, streamlining protein purification, and allowing an easy way to track proteins during protein expression and purification.
A wide variety of protein purification methods that can be combined to generate a suitable purification scheme are available. Usually, one executes a series of purification steps, and only rarely proteins can be purified in a single step.
Early steps combine low-resolution and high-capacity methods at later stages of purification scheme. For low-resolution protein purification, methods such as fractional precipitation and two-phase partition systems usually employed. For applications requiring the highest purity and relatively small Protein Purification amounts of protein, chromatography can be chosen to selectively purify the target protein.
There are two stages of protein production in cells: Transcription and Translation, which is known as the central dogma of molecular biology. In other words, Transcription and Translation steps belong to the recombinant protein expression steps.
To make recombinant proteins, the gene is isolated and cloned into an expression vector. Generating a recombinant protein requires the protein expression system, protein purification system and protein identification systems.
Basic steps to get recombinant Protein:
1. Amplification of gene of interest.
2. Insert into cloning vector.
3. Sub cloning into expression vector.
4. Transformation into protein expressing host (bacteria (E coli), yeast, mammalian cells or baculovirus-insect cell system).
5. Test for identification of recombinant protein.( Western blot or Fluorescence)
6. Large scale production. (Large scale fermentation)
7. Isolation and purification.
There are various factors to be considered:
1. Which host system to be selected?
2. How to isolate and purify the recombinant proteins?
It is not easy to select a proper expression host or to use the right purification method, where the properties of the interested recombinant protein should be considered. Some of the important factors are listed below:
• Membrane bound
• Single or multidomain
• Size (molecular weight)
• Where it expressed
Recombinant protein production is time-consuming for most of those who have not enough experience to express and isolate a recombinant protein. There are many biological companies supply good service for various types of recombinant protein expression in different scales such as Sino Biological Inc. (See the detail service list of recombinant protein production)
Yilmaz H, et al. (2019) Production of recombinant n protein of infectious bronchitis virus using the baculovirus expression system and its assessment as a diagnostic antigen. Appl Biochem Biotechnol 187(2): 506-517.
Zhu J (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30(5): 1158-1170.
Labrou NE (2014) Protein purification: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 3-10.