The Detailed Construction Method of recombinant protein

Recombinant protein is produced by transferring the target gene to a vector and expressed in host cells. It plays important roles in signal transduction, pharmaceutical development and scientific research. Below is an introduction on production process of recombinant proteins.

1.Gene amplification

（1）Primers synthesis

Primers are designed by analyzing the gene sequence as well as the protein structure. Trans-membrane area and complex secondary structures should be avoided. Meanwhile, it is preferred to choose N or C terminal sequence and the evaluation of the primers should be high. Search restriction sites and proper vector.

（2）Gene cloning and identification of recombinant colonies.

1）PCR

The RNA in specific tissue where target protein expresses is used for PCR amplification. Target fragment is obtained by gel recovery after agarose gel electrophoresis. After a repeated amplification by PCR, a large amount of cDNA can be obtained.

2）Restriction enzyme digestion and ligation

Target gene fragment and vector are digested by the same restriction enzyme, after gel-purify, insert fragment and vector are ligated by ligase.

3）Transformation and identification

The ligased products are transferred to competence E.coli and cultured in LB plate overnight. Pick the colonies on the plate for PCR detection. Then correct coloniesare identified by agarose gel electrophoresis and sequenced. Correct strains arestocked for further use.

2.Protein expression

（1）Small scale expression

Appropriate strain is inoculated into the medium and expression of the target gene is induced by the addition of IPTG. Identify the expression of target gene by SDS-PAGE. Large scale expression will be carried out if the target protein can successfully express.

（2）large scale expression

400-800 ml culture medium are used for large scale expression. Identify the expression by the molecular weight of target protein,using SDS-PAGE. If protein can be successfully expressed, larger scale expression is carried out using 800-1600 ml culture medium. Evaluate the expression by SDS-PAGE.

3. Protein purification

Nickel column is used for protein purification if the vector contains his-tag. Several proteins may form inclusion body. We have introduced the procedure in the previous topic Purification of recombinant protein expressed in E. Coli 

The molecular weight and purity will be identified by SDS-PAGE, BCA, etc. Regularly the purity is over 95%.

4.Quality Control

The purified proteins will be dried out and common tests are carried out to verify the quality of the protein.

Recombinant proteins are produced by the principle and procedures mentioned above. Proteins produced by prokaryotic system is taken as an example. Each step is essential during the production process, and different proteins will face differnt difficulties, including the amplification of target gene, the formation of inclusion body etc. Cloud-Clone Corp. have many years experiences in producing recombinant proteins with prokaryotic ,eukaryotic and mammalian expression systems. We can also offer custom service which can reach the specific requirement of customers.

For more information, please visit: http://www.cloud-clone.us/.