Enzyme Kinetics with ?-glucosidase
The lab report for the BM327 Biochemistry class will focus on Laboratory 3 (Enzyme assays with ?-glucosidase). Students will also be expected to apply knowledge from other laboratory sessions (Lab 12, Virtual Protein Purification; Lab 4, Functionality of Proteins) to outline a procedure for the purification of recombinant human ?-glucosidase enzyme.
The lab report should have 4 sections as described below and the weighting for each section is indicated. Details about content and organisation/structure of each section are outlined below.
1. Abstract: Students should apply knowledge gained in the assessment of BM327 Biochemistry in semester 1 to prepare an abstract for the ?-glucosidase lab. The abstract should include a brief introduction and aim of the laboratory, a brief description of methodology, description of key results obtained, and conclusions/interpretation of data (if data is not as expected this should be noted in the abstract). The maximum word count for the abstract is 250 words.
2. Results: In the Results section you should present your results in an appropriate form (i.e. graphs and tables). Ensure that all graphs have appropriate x and y axis titles. In the laboratory, you undertook 8 different reactions, and the data from these reactions should be presented as follows:
a. Figure 2.1 should show data from reaction 1.
b. Figure 2.2 should be a single graph that shows data from reactions 2 and 3 (reproducibility).
c. Figure 2.3 should be a single graph showing reaction 4 (increased enzyme), reaction 5 (increased substrate) and either reaction 2 or 3 as the control (your choice between reactions 2 and 3 should reflect which dataset you think is the most accurate).
d. Figure 2.4 should be a single graph showing reaction 6 (increased pH) and either reaction 2 or 3 as a control.
e. Figure 2.5 should be a single graph showing reaction 7 (boiled enzyme), reaction 8 (inhibitor) and either reaction 2 or 3 as control.
f. Finally, you should include a table showing the initial rates for each reaction (remember to use the correct units) and also indicating the data points that were used to calculate the initial rates.
As you are showing multiple datasets on the same graph, make sure that you design the graphs such that each dataset is clearly distinguishable from the others.Each figure needs to have a figure legend. The legend should include the figure number, figure title, and a description of the figure content.As all experiments only had a single sample per data point, do not include statistical analyses. The figures/tables in this section should be accompanied by text that briefly describes the procedure followed and the results obtained, including initial rates for each reaction (do not interpret your results in this section, just present them). The maximum word count for this section is 500 words (not including figure legends).
3. Discussion: In the Discussion section you should discuss your findings. You should focus on the following issues:
a. Explain why measurements for reaction 1 were different from reactions 2 and 3. Can you determine the expected level of p-nitrophenol ionisation at pH 5.5 versus pH 9.2 (note: you need to apply a specific equation to do this)?
b. How was reproducibility of the results (compare reactions 2 and 3)? If reproducibility was poor, what are the possible reasons?
c. How did the initial rates for reactions 4-8 compare with those of reactions 2-3? You should state here what you found for each reaction and whether this was expected or not.
If any results were unexpected discuss possible reasons for this. Make sure that you clearly discuss why you would expect the rate to be different in reactions 4-8 compared with reactions 2 and 3. The maximum word count for this section is 500 words.
4. Future work: You are tasked with producing human lysosomal?-glucosidase also called glucosylceramidase(protein id: NP_000148.2 glucosylceramidase isoform 1 precursor) for use as a remedy against Gaucher disease.For obvious reasons this protein cannot be produced from human tissue and requires to be generated in a recombinant form of the enzyme. You have the plasmidpSF-CMV-Puro-COOH-TEV-GST available for cloning and expressionof ?-glucosidase as a COOH-terminal glutathione-S-transferase (GST) fusion protein in Chinese hamster ovary cells (CHO)(see BM327 Biochemistry on Myplacefor the sequence of the plasmid and its map below). Remember to include a figure legend for each figure.
a. Examine the properties of human lysosomal beta-glucosidase (use SPOCTOPUS for the identification of a signal sequence for ER import; EXPASY tool forisoelectric point and molecular mass of the mature protein;NetNGlyc 1.0 for N-glycosylations). Display the protein sequence in your report and highlight sites for modifications and signal sequences[Fig. 4.1]. Use the appropriate software (see manual BM327 Biochemistry Lab4) for in silico analyses and back up your findings by evidence from the scientific literature (include citation).
b. Produce a plasmid map of pSF-CMV-Puro-COOH-TEV-GSTusing the sequence provided on Myplace and Serial Cloner (several tutorials for Serial Cloner are available on YouTube) [Fig. 4.2].
c. Identify the open reading frame (ORF) from the mature mRNA provided below (NM_001005741.2 Homo sapiens glucosylceramidase beta (GBA), transcript variant 2, mRNA). Show mRNA sequence with ORF and stop codon in a different colour [Fig. 4.3].
d. Introduce HindIII and XhoIrestriction enzyme cleavage sites at the 5′- and 3′-end of the ORF, respectively ensuring that they allow cloning in-frame with the ORF of GST. Check that the sites you have introduced are not present within the ORF sequence (if so, change the internal sitestaking care that the change does not lead to a change of the encoded amino acid; silent mutation). Highlight any aIDitions and changes in the sequence of the ORF and display as figure [Fig. 4.4].
e. Use Serial Cloner to insert the manipulated ORF into pSF-CMV-Puro-COOH-TEV-GST ensuring the number of aIDitional amino acids introduced for the cloning is minimaland generate a plasmid map for the expression construct. Only show those restriction sites that were relevant for inserting the beta-glucosidase ORF into the plasmid (untick “Unique Sites” and “Double Site” and tick “Particular Sites” and choose the relevant ones from the drop down list). Use the same colour code for your plasmid as for the plasmids shown below and display the plasmid as a figure [Fig. 4.5].
f. Show the relevant part of the restriction map copied from Serial Cloner that proves in frame ligation of the beta-glucosidase gene with GST[Fig. 4.6].
g. The fusion protein will be secreted by the CHO cells. Hence, it can be purified from the cell culture supernatant using reversible binding to glutathione-sepharose followed by cleavage with TEV protease to remove GST. TEV protease cleaves the fusion protein between glutamine and glycine of the ENLYFQG recognition sequence. Display the amino acid sequence of the mature beta-glucosidase after TEV cleavage and calculate its molecular mass and isoelectric point[Fig. 4.7].
h. Devise an ion exchange chromatography method (ion exchange material, pH in purification buffer) that would allow further purification of the proteinafter TEV cleavage assuming that you want to bind the protein to the ion exchange material and then elute it with a salt gradient.List the following:
(i). pH in purification buffer
(ii). Charge of protein at that pH (positive or negative)
(iii). Type of ion exchange material to be used also indicating its charge
(iv). Name an example of a possible ion exchange material
Note: Under normal circumstances beta-glucosidase is a lysosomal protein.
>NP_000148.2 glucosylceramidase isoform 1 precursor [Homo sapiens]
MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQ PEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPIDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTS PTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLIDQRLLL PHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNW VRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ
>NM_001005741.2 Homo sapiens glucosylceramidase beta (GBA), transcript variant 2, mRNA
Aim: To set up an enzymatic assay using different assay and detection conditions.
This practical is of importance for students studying Biochemistry as enzymes play important roles in all cells, organs and organisms. Enzymatic characterisation helps to understand the function and properties of a protein irrespective of its origin, microbe or immune cell.
At the end of the practical you should be able to:
– Understand how to carry out a specific enzymatic assay.
– Understand the effect of pH, substrate and enzyme concentration on an enzymatic reaction.
– Understand the concept of enzyme inhibition and inactivation.