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RECOMBINANT HUMAN tPA AS A DRUG Tissue plasminogen activator (tPA) is a naturally occurring protein that cleaves the inactive zymogen plasminogen into the active enzyme plasmin. Plasmin then cleaves fibrin clots. This process is important in removing clots after the repair of a wound has been completed. Clots often form in response to disease conditions such as atherosclerosis, stroke, cancer and some complications during pregnancy. Doctors now use recombinant tPA as a powerful drug to help dissolve these clots and prevent much of the damage associated with loss of blood flow to vital organs. In this section we will examine how tPA was cloned, expressed and modified to create this cruical drug. We will be using information from several articles to understand different techniques used in molecular biology research. |
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The first five sections contain materials from a Nature article describing the initial cloning of tPA and its expression in bacteria. Identification of the tissue producing tPA Production of a cDNA library containing tPA. Screening a cDNA library for the tPA cDNA, and DNA sequencing Screening a genomic library for the tPA gene and sequence analysis
The next article focuses on expression of tPA in yeast
The final articles examine expression in mammalian cells and in vitro mutagenesis
Identification of the tissue producing tPA 1. Why would we want to isolate mRNA when cloning a gene? Why not work directly with genomic DNA (Hint: What is different about mRNA and genomic DNA)? 2. How was 35S methionine used to determine which cells were producing tPA? Why did the authors need to know which cells were producing t-PA? (Fig. 1) 3. How was an oligo(dT) cellulose column used to purify mRNA? (Fig. 2A) 4. How was the mRNA from these cells fractionated by size? Would lane 10 contain smaller or larger mRNA molecules than lane 2? Why? (Fig. 2A) 5. How did the authors use in vitro translation to determine which fractions contained the tPA mRNA? Why was immunoprecipitation necessary? (Fig. 2A)
Production of a cDNA library containing tPA. 1. How did the authors produce complementary DNA (cDNA)? What enzyme was necessary? (text) 2. How was the DNA introduced into the pBR322 plasmid? What enzymes were necessary? 3. Draw a picture of the steps involved in making the library.
Screening a cDNA library for the tPA cDNA, and DNA sequencing 1. How was a DNA probe designed to screen the library? What is degenerate DNA? (text) 2. How were colonies containing the tPA cDNA identified? 3. How was DNA sequencing used to confirm the presence of the tPA cDNA? 4. Was the cDNA full length, or was part of the tPA coding region missing? How did the authors know this? Hint: how many amino acids are in human t-PA, and how many were in the clone?
Screening a genomic library for the tPA gene and sequence analysis 1. Why was a genomic library used to screen for tPA? (text) 2. How was the genomic library different from the cDNA library? Why was a virus used instead of plasmids to hold the genomic DNA? 3. What was used as a probe to screen the genomic library? 4. How was the clone obtained from the genomic library used to get the amino terminal region of tPA? 5. What is a Southern blot? What was used as a probe? 6. How was the Southern blot used to determine the number of tPA genes in the genome? Why was this important to determine? 7. Examining the restriction map of the cDNA, (Figure 3A), how did the authors explain the results of the Sourhtern Blot? (Figure 3C - note: this is simulated data based on the values given in the text as ``data not shown"). 8. Did the two cDNAs overlap, or was part of the tPA coding region missing? How did the authors know this? (Fig. 3A) 9. What structural features of a gene were the authors able to identify in the final cDNA sequence? (text)
Cloning and expression of the tPA cDNA in E. coli. 1. The authors faced a problem when they decided to express human tPA in bacteria because the cDNA was in two fragments. They used the strategy outlined in figure 4 to attach the two fragments together. Restriction Map Exercise 2. Why was the enzyme HhaI important? (text) Why was the enxyme Sau3A used? (Hint: what was deleted with this enzyme). 3. Two synthetic oligonucleotides were used to alter the 5´ end of the cDNA. Identify and explain three important features of these oligonucleotides (what did they need to contain)? 4. The oligos and two fragments at the 5´ endo of the cDNA were joined with ligase and then digested with EcoRI, NarI and BglII. Why was this step done? 5. In the last step two fragments were simultaneously ligated into a new plasmid for expression in bacteria. How did the authors design the reaction so that the fragments could only be inserted into the plasmid in the correct orientation? 6. In the end the authors reported an activity of 3-5 U per A550 (~1 L of culture). They also reported about 50-80 ug of tPA protein per L of culture. How did their activity correlate with published values of tPA? What percent is the specific activity of the recombinant protein relative to the human protein? How do the authors explain any differences? Could you think of other possible explanations? 7. Recently a group has reported the production of active tPA in bacteria that are also expressing protein disulfide isomerase. In light of this work could you think of other explanations for the differences in specific activity observed in question 6? Expression of active human tissue-type plasminogen activator in Escherichia coli. Qiu J, Swartz JR, Georgiou G. Appl Environ Microbiol 1998 Dec;64(12):4891-6 While tPA could be made in bacteria its activity was very low. Researchers also tried yeast, a simple eukaryotic expression system, to see if active tPA could be made cheaply. Expression of tPA in yeast 1. What is a leader sequence? Why do the authors want to fuse the t-PA cDNA with a yeast leader seqeunce? What leader sequence did they choose? 2. In the cloning of the t-PA cDNA with the leader sequence was it important to pay attention to the reading frames of each peptide? What two other sequences did the authors need to be sure to include? 3. What is a promoter? What is an inducible promoter? Which inducible promoter did the authors use? How did they induce the promoter? 4. Examining Fig. 2, which fractions appeared to contain the most t-PA? Which contained small amounts of t-PA? None? 5. How did the authors purify the t-PA? Why would it be preferable to have the protein secreted? What was the final specific activity? Was it the same as purified human t-PA? Was it the same as recombinant t-PA expressed in E. coli?
Mammalian proteins are usually best expressed in mammalian cells. These cells perform all of the necessary secondary modifications and recognize signal peptides, allowing proteins to be secreted.
Making a good thing beter: In vitro mutagenesis Often specific properties of a protein can be altered by changing select amino acids. Making changes in amino acids can also be used to identify the function of regions of a protein. Specific amino acid changes are produced by site-directed mutagenesis of the DNA followed by expression of the mutant protein. 1. In the first paper the authors use alanine scanning mutagenesis to identify functional regions of t-PA. What is this technique and which amino acids did they focus on. Why? 2. What was the goal of this mutagenesis study, i.e. what activities were the authors trying to alter? 3. How many mutants were made? Why was single stranded DNA used? 4. What type of cells were used to express the mutant proteins? What is meant by transient expression? Why isn´t expression permanent? 5. In the second paper the authors have introduced three different mutants into one protein. What type of cells were used to express the mutant proteins? How was stable expression generated? 6. How did they choose these mutants? What was the affect of each change?
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