A large biotechnology has subcontracted your services to produce human lactate d
ID: 68216 • Letter: A
Question
A large biotechnology has subcontracted your services to produce human lactate dehydrogenase. You decide to use DNA extracted from human fibroblasts to insert the gene into a bacterial plasmid. The lactate dehydrogenase gene has a length of 20417 base pairs. There are several introns in the gene. The exons are as follows Base pairs that form exons: Moreover, on this sequence, the open reading frame spans from the position 7409 to 17847 on the DNA. What is the length of the messenger RNA (excluding the poly(A) tail)? What is the length of the polypeptide chain (number of amino acids prior to any posttranslational modifications)? You decide to produce your enzyme in E. coli using recombinant DNA. To facilitate the purification of the enzyme, you add a 5' signal peptide to the cloned sequence to allow the secretion of the protein. You measure the enzyme activity both in the medium and in lysed cells and you notice that there is almost no secreted enzyme. What is the most likely cause for the lack of secretion of the enzyme into the medium? You observe a contamination in your medium. At the same time, you notice that the pH of the medium is much lower than usual. Based on the metabolism of different microorganisms, explain what metabolic pathways and molecules may cause this decrease in pH. Lactate dehydrogenase catalyzes the conversion of pyruvate into lactate. Write the complete balanced reaction of pyruvate conversion to lactate using this enzyme. Calculate the standard Gibbs free energy change of the reaction using the reference charts provided on myCourses. Calculate the equilibrium constant for this reaction. You perform an enzyme kinetics experiment. What is the equilibrium concentration of lactate if the reagents used are 2 mM pyruvate and 0.5 mM lactate?Explanation / Answer
1. a) The length of mRNA is 994bps.
b) The length of polypeptide chain is =994/3 = 331.3 aminoacids
2. Usually, the signal peptide compatible to other organisms might not be compatible in E.Coli. So, it is necessary to use the signal peptide that is specific to E.Coli. It is also observed that E.Coli is not is not suitable for the functional expression of proteins of peripasmic region.
3. Carbohydrate metabolism in the microorganisms is found to lower the pH of the medium.
4. a) Pyruvate + NADH + H+--------------------> Lactate + NAD+ is catalyzed by lactate dehydrogenase.
The standard Gibb’s free energy change is -25.1 kJ/mol.
b) and c) The data provided is not sufficient to calculate the equilibrium constant and in turn the equilibrium concentration of lactate. Temperature at which the reaction occurs has to be provided to solve the equation,
Keq = exp (-G'/RT). If Keq is not calculated, the concentration of lactate cannot be determined.
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