Producing Human Proteins in E Coli

Once the above operations are finished, it is necessary to make many copies of the pieces of recombinant DNA. Copying uses the natural copying mechanism of live bacteria by introducing the recombinant DNA molecule that was made in the test tube into bacterial cells, generally Escherichia coli (E. coli). When treated with appropriate chemicals,

Figure 5.3 Making Human Insulin Using Recombinant DNA. A. Partial amino acid sequence of human insulin. B. The DNA sequence corresponding to the amino acid sequence shown in A. C. Promoter and terminator suitable for high-level transcription in E. coli are added. D. The insulin gene with the promoter and terminator is flanked by restriction enzyme sites that correspond to the restriction enzyme used to cut the plasmid. E. Plasmid DNA is cut with the restriction enzyme. F. Pieces of DNA with the insulin gene and the cut plasmid DNA join spontaneously at their complementary ends. Finally, an enzyme is used to glue the sugar-phosphate backbones of the two DNA pieces. The * indicates where the ligase enzyme glues the sugar phosphate backbone on the recombinant DNA. This process results in a recombinant DNA molecule containing the human-insulin gene in a bacterial-plasmid DNA. The engineered human-insulin gene is shown as the straight, short double helix, and plasmid DNA is shown as a curved double helix.

Figure 5.3 Making Human Insulin Using Recombinant DNA. A. Partial amino acid sequence of human insulin. B. The DNA sequence corresponding to the amino acid sequence shown in A. C. Promoter and terminator suitable for high-level transcription in E. coli are added. D. The insulin gene with the promoter and terminator is flanked by restriction enzyme sites that correspond to the restriction enzyme used to cut the plasmid. E. Plasmid DNA is cut with the restriction enzyme. F. Pieces of DNA with the insulin gene and the cut plasmid DNA join spontaneously at their complementary ends. Finally, an enzyme is used to glue the sugar-phosphate backbones of the two DNA pieces. The * indicates where the ligase enzyme glues the sugar phosphate backbone on the recombinant DNA. This process results in a recombinant DNA molecule containing the human-insulin gene in a bacterial-plasmid DNA. The engineered human-insulin gene is shown as the straight, short double helix, and plasmid DNA is shown as a curved double helix.

E. coli cells can take up DNA molecules in their growth medium. Once inside the bacterial cells, the recombinant DNA molecules can be copied, just like plasmids naturally found in E. coli. In fact, the transformed E. coli cells do not "know" that the plasmids now contain human DNA, and they replicate it along with the plasmid sequences to which they are joined. This is why plasmids used in cloning are called vectors; they are used to ferry pieces of foreign DNA into bacterial cells.

As we just saw, E. coli cells can make hundreds of copies of recombinant plasmids containing a human gene. This means that E. coli in the laboratory contains hundreds of copies of the human gene you want to express, or, in other words, the dosage of human genes in these bacterial cells is very high. With the appropriate promoter and terminator, these human-gene copies are transcribed at high level (many mRNA molecules are made), and consequently, translation of these mRNAs will generate large amounts of proteins corresponding to that gene. This is possible thanks to the fact that the genetic code is universal. In fact, not only does E. coli not "know" that it harbors and actively replicates a piece of human DNA, it is also "unaware" that it is making large amounts of human mRNA and large amounts of the corresponding human protein. Today, large vats, called fermenters, are used to grow engineered E. coli on an industrial scale. The protein of interest is purified from these cells and sold by pharmaceutical companies.

Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook


Post a comment