How to express a protein with bioactivity?


View:1358 Time:2017-08-15


Protein production - a process in which a specific protein is produced -- has become an extremely important biotechnology in biological and biomedical science. In most cases, the protein of interest has a certain form of bioactivity in vivo. Depending on the application of the protein produced, we may put different requirements on its bioactivity. But how can we ensure that the protein is produced with bioactivity? This article will focus on this topic and offer some solutions.

Firstly, choosing an appropriate expression system is required.

After many years of development, a variety of expression systems has been created, which can usually be categorized into either cell-based or cell-free systems. Cell-based systems can be divided into bacterial systems which include Escherichia coli, Corynebacterium, and Pseudomonas fluorescence, and eukaryotic systems, which include yeast (Saccharomyces cerevisiae, Pichia Pastoris), Filamentous fungi, Baculovirus-infected cells, Non-lytic insect cell expression, Leishmania and Mammalian systems. Choosing a suitable one from so many different expression systems is somewhat difficult because different expression systems may have different characteristics. For example, E. coli is a type of prokaryotic organism that lacks post-translational modification mechanisms such as glycosylation modification, so it is usually not a good choice for expression of complex eukaryotic proteins.

Please visit here to read a table and know more the advantages and applications of five commonly used expression systems. It will help you to choose an appropriate expression system.

Secondly, choosing an appropriate vector is required.

A vector is a DNA molecule that helps deliver foreign genetic material into another cell, where it can be replicated and/or expressed. Cloning vectors is a useful method to produce many copies of your gene of interest. Expression vectors have an impact on the actual expression of the gene into mRNA and protein in the target organism. Among the four major types of vectors-plasmids, viral vectors, cosmids and artificial chromosomes, plasmids are used most frequently. There are a series of available plasmid vectors, but how to choose the best one? A lot of factors influence the choice of vector, such as insert size, copy number, incompatibility, a selectable marker, cloning sites, and specialized vector functions. Here is a table of commonly used vectors.

Table 2 Commonly Used Vectors of Cusabio

Expression System

Vectors

Tag

E. Coli Expression System

pGEX-6p-1

N-terminal GST-tagged

pGEX-4T-1

N-terminal GST-tagged

pGEX-4T-2

N-terminal GST-tagged

pET21a(+)

C-terminal 6xHis-tagged

pET21b(+)

C-terminal 6xHis-tagged

pET22b(+)

C-terminal 6xHis-tagged

pET-23b(+)

C-terminal 6xHis-tagged

pET-26b(+)

C-terminal 6xHis-tagged

pET28a(+)

N-terminal 6xHis-tagged and C-terminal 6xHis-tagged

pET-29a(+)

C-terminal 6xHis-tagged

pET30a(+)

N-terminal 6xHis-tagged and C-terminal 6xHis-tagged

pET32a(+)

N-terminal 6xHis-Trx-tagged and C-terminal 6xHis-tagged

pET-43.1a(+)

N-terminal 6xHis-NusA-tagged and C-terminal 6xHis-tagged

pMal-c2X

N-terminal MBP-tagged

pColdIII

NO-tagged

pBV220

NO-tagged

pACYCDuet-1

N-terminal 6xHis-tagged

pETDuet-1

N-terminal 6xHis-tagged

Yeast Expression System

pPIC9K

NO-tagged

pPIC3.5K

NO-tagged

pPICZα A

C-terminal Myc-tagged and C-terminal 6xHis-tagged

pGAPZα A

C-terminal Myc-tagged and C-terminal 6xHis-tagged

pPICZ A

C-terminal Myc-tagged and C-terminal 6xHis-tagged

pPinkα-HC

NO-tagged

pPinkα-LC

NO-tagged

Mammalian Cell Expression System

pCMV6-Entry

C-terminal Flag-tagged

pSecTag2A

C-terminal 6xHis-tagged

pTT5

NO-tagged

Insect baculovirus expression system

pFastBac 1

NO-tagged

pFastBac HTB

N-terminal 6xHis-tagged

pFastBac Dual

NO-tagged

Cell free expression system

pET21a

N-10xHis tag

pET28a-sumo

N-10xHis tag-sumo tag

pET23b

C-6xHis tag

When you choose a vector, youd better take these things into considerations:

Insert size

The only aspect to consider here is whether you are cloning a large or small DNA fragment. Vectors must be relatively small molecules for a convenience of manipulation. Too large vectors may affect the replication and cause problems with stability. Usually, plasmids can cope with inserts up to 15 kb. But there are also special plasmids that cope with larger inserts.

Selectable marker

Choose a selectable marker is required. A marker allows for the identification of a positive transformant. There are two major types of selectable markers: drug-resistance markers (which involve the incorporation of a gene encoding an enzyme that inactivates a specific antibiotic) and auxotrophic markers (which allow cells with the marker to survive without essential nutrients in the medium).

Restriction site

There must be multiple convenient restriction sites that can be used for insertion of the DNA to be cloned.

Copy number

Generally, a high-copy plasmid vector is better. But in some cases, a low-copy vector is needed because high-copy plasmids may cause problems like toxicity.

Thirdly, choosing an appropriate purification method is required.

For some applications, a crude extract is sufficient. However, for other use, a high level of purity is required. To produce the protein with high purity, purifying the protein produced is necessary. Protein purification refers to a process that isolates one or more proteins from cells, tissues or whole organisms. A variety of purification strategies has been developed, such as Size exclusion chromatography (SEC), Separation based on charge or hydrophobicity, Affinity chromatography (AC), and HPLC.

Fourthly, validation experiments are important.

To confirm the bioactivity of the protein produced, doing a validation experiment is usually required. The folding, modification and activity should be measured. The biological activity of a recombinant protein is routinely measured using a bioassay such as an enzyme assay. Enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) are two of the most powerful methods for detecting a particular protein in a complex mixture.

ELISA is a method that uses antibodies and color change to identify substances that have antigenic properties, such as proteins, hormones, bacterial antigens, and antibodies. ELISA tests are generally highly sensitive and specific.

WB is a technique used to identify and locate proteins based on their ability to bind to specific antibodies. It can give you information about the size of your protein (with a comparison to a size marker or ladder in kDa), and also give you information on protein expression (with a comparison to a control such as untreated sample or another cell type or tissue).

In conclusion, expression of recombinant proteins with bioactivity is a complex process that many aspects should be taken into considerations. As a manufacturer, Cusabio offers protein expression service to help you do your research. More details about Cusabio protein service, please visit https://www.cusabio.com/protein_service/

 

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