MCB 2050 Lecture Notes - Lecture 5: Globulin, Protein Tag, Northern Blot

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bluehyena388

23 Jan 2018

School

University of Guelph

Department

Molecular and Cellular Biology

Course

MCB 2050

Professor

Dick Mosser

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Document Summary

**techniques of molecular genetics (snustad 6e: ch 14)*: techniques for protein analysis. They are the end products of gene expression: measuring changes in protein levels is more relevant than measuring changes in mrna levels. Protein function is what controls the actions of cells: metabolism, gene expression, dna replication, cell movement, cell signaling, cell division, differences in protein content defines the activities of different cell types. Disease states are the consequences of deregulated protein expression or function. In order to understand the molecular basis of a disease it is necessary to understand what protein is responsible and how its function is deregulated: level of expression. Cells are lysed in buffer containing the detergent sodium dodeycl sulfate. Sds binds proteins imparting a net negative charge, migrates to anode. Polypeptides migrate through a porous polyacrylamide gel matrix in an electric field. Mobility is a linear function of the logarithms of their molecular mass (smallest polypeptide runs fastest)

Related Questions

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; âprotein purification.pptâ

4. What method did we use to attempt to purify your LepA/EF 4 â GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is âProtein Aâ and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why canât we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30Â°C VS. 37Â°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-FâlacI^Q -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacI^Q, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacI^Q gene contains a âpromoter-upâ mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (MÃ¼ller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of âleakyâ expression from the lac promoter. It was shown by comparing two identical strains (one contained lacI^Q and one contained lacI) that pPEM109 could only be maintained in the lacI^Q containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

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DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg²⁺ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

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DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

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DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 â GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 â GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP â binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 â GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 â GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 â GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP â specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 â GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 â GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

*Please don't answer with photos, DO NOT UPLOAD PHOTO, it's hard to read.

I post this question twice, please don't answer this post if you already answer the other one, if you can answer different answer that's fine. REASON WHY I POST IT TWICE, I NEED TWO DIFFERENT VIEW.

Discussion

Hi All - this week you will learn about DNA - the molecule of life! You may think that protein-coding genes are the most important, but results from the Human Genome Project revealed that only about 2% of our DNA codes for protein - so what about the rest of the "junk DNA" (or non-protein coding portion of the genome) - is it functionally important? Yes! Scientists are learning how "the non-protein-coding portion of the genome is of crucial functional importance: for normal development and physiology and for disease". I would like you to please choose a topic with regards to the non-protein coding portion of the genome (this will introduce you to gene regulation which we will cover in chapter 11) or you may also choose to write about a topic that describes a mutation in any protein that is involved in DNA replication, transcription, or translation (we did not talk about all of the enzymes involved in those processes). Please list your reputable source - this is worth 0.5 pt (even if you use one of my sources below) and tell me WHY you chose your topic (this is worth 1 pt.) please try to briefly explain your topic to the best of your knowledge - I don't expect you to write a lot of detail about your topic - it might be hard to understand (which you are welcome to comment on in your paragraph)!

Examples to choose from (but not limited to) include: noncoding RNAs (snRNAs, miRNAs, siRNAs, snoRNAs, piRNAs, lincRNAs) and RNA therapeutics:

LIST:

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119351/ (Development of microRNA therapeutics is coming of age)

2. http://www.umassmed.edu/rti/ (RNA Therapeutics Institute)

3. http://www.genengnews.com/insight-and-intelligence/rna-based-therapeutics-and-vaccines/77900520/

4. http://genesdev.cshlp.org/content/26/21/2361.full (Biology of PIWI-Interacting RNAs: new insights into biogenesis and function inside and outside of germlines)

5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265212/ (PIWI proteins and their interactors in piRNA biogenesis, germline development and gene expression)

6. http://www.news-medical.net/news/20121203/microRNAs-play-an-important-role-during-embryonic-development.aspx

7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578617/ (the role of microRNAs in cell fate determination)

8. http://news.berkeley.edu/2017/01/13/scientists-reprogram-embryonic-stem-cells-to-expand-their-potential/

9. http://www.the-scientist.com/?articles.view/articleNo/40871/title/The-Second-Coming-of-RNAi/

10. http://web.mit.edu/newsoffice/2013/cardiac-development-needs-more-than-protein-coding-genes-0124.html (long non-coding RNA molecules)

11. http://www.alzforum.org/news/research-news/new-role-micrornas-torpedoes-sink-neurons(microRNAs)

12. http://ghr.nlm.nih.gov/condition/rothmund-thomson-syndrome (mutation in human helicase gene)

13. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633371/ (mutation in mitochondrial helicase gene called TWINKLE)

14. http://sfari.org/news-and-opinion/news/2013/in-autism-related-disorders-rna-turns-out-to-be-key (RNA topoisomerase)

15. http://ghr.nlm.nih.gov/gene/TERC (dyskeratosis congenita - mutations in the TERC gene - the RNA component of telomerase)

16. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853347/ (Top3beta is an RNA topoisomerase that works with Fragile X syndrome protein to promote synapse formation)

The following 3 articles discuss long non-coding RNAs - there are many examples in each article so you only need to talk about one example - not the whole article:

1. http://www.sciencedirect.com/science/article/pii/S2468054016300063 (mechanism of action and functional utility)

2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084630/ (dysregulated expression in ovarian cancer)

3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736767/ (stem cells)

skyminnow948

Enzyme Dysfunction in Disease (30 pts)

Protein kinases are key regulators of cellular physiology and, consequently, their dysfunction is associated with many diseases. Conversely, mutations and other defects sometimes help to illuminate gene function. Continuing with the exploration of the PI3K/AKT pathway, this exercise will seek to illuminate the role of certain residues and motifs in molecular functioning of AKT.

(1) Diseases associated with AKT1 mutations were identified in the first exercise. Following the same procedure and PUBMED searches, identify diseases associated mutations of AKT2 and AKT3 (some relevant papers are provided below).

(2) Through literature search or the references below, generate a table of disease-associated mutations of the above genes.

(4) Generate a hypothesis on the role of the PI3K/Akt in cancer or any other disease.

(5) Make predictions based on experiments that can be conducted in cell lines.

(4) Conduct sequence alignments of these proteins by selecting homologues from divergent species and identify the most highly conserved residues and motifs.

(5) Download the structure of AKT with and without ligands from the PDB and propose the consequences of the above mutations in protein function.

(6) Propose some experiments to test the predictions.

(7) Highlight three of the most highly conserved motifs on the protein structure and comment on their possible role in AKT function.

1. Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery.

Karve S, Werner ME, Sukumar R, Cummings ND, Copp JA, Wang EC, Li C, Sethi M, Chen RC, Pacold ME, Wang AZ. Proc Natl Acad Sci U S A. 2012 May 22;109(21):8230-5. doi: 10.1073/pnas.1120508109. Epub 2012 Apr 30. PMID: 22547809 Free PMC Article

2. Phase I clinical, pharmacokinetic, and pharmacodynamic study of the Akt-inhibitor triciribine phosphate monohydrate in patients with advanced hematologic malignancies.

Sampath D, Malik A, Plunkett W, Nowak B, Williams B, Burton M, Verstovsek S, Faderl S, Garcia-Manero G, List AF, Sebti S, Kantarjian HM, Ravandi F, Lancet JE. Leuk Res. 2013 Nov;37(11):1461-7. doi: 10.1016/j.leukres.2013.07.034. Epub 2013 Aug 6.PMID: 23993427

The Mechanism of ATP-Dependent Allosteric Protection of Akt Kinase Phosphorylation.

Lu S, Deng R, Jiang H, Song H, Li S, Shen Q, Huang W, Nussinov R, Yu J, Zhang J.

Structure. 2015 Sep 1;23(9):1725-34. doi: 10.1016/j.str.2015.06.027. Epub 2015 Aug 6.

PMID:26256536

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Select item 262867482.

Alternative activation mechanisms of Protein Kinase B trigger distinct downstream signaling responses.

Balzano D, Fawal MA, Velazquez JV, Santiveri CM, Yang J, Pastor J, Campos-Olivas R, Djouder N, Lietha D.

J Biol Chem. 2015 Aug 18. pii: jbc.M115.651570. [Epub ahead of print]

PMID:26286748

Free Article

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Mutant AKT1-E17K is oncogenic in lung epithelial cells.

De Marco C, Malanga D, Rinaldo N, De Vita F, Scrima M, Lovisa S, Fabris L, Carriero MV, Franco R, Rizzuto A, Baldassarre G, Viglietto G.

Oncotarget. 2015 May 25. [Epub ahead of print]

PMID:

26053093

Free Article

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Activating AKT2 mutation: hypoinsulinemichypoketotic hypoglycemia.

Arya VB, Flanagan SE, Schober E, Rami-Merhar B, Ellard S, Hussain K.

J ClinEndocrinolMetab. 2014 Feb;99(2):391-4. doi: 10.1210/jc.2013-3228. Epub 2013 Nov 27.

PMID:

24285683

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AGC protein kinases: from structural mechanism of regulation to allosteric drug development for the treatment of human diseases.

Arencibia JM, Pastor-Flores D, Bauer AF, Schulze JO, Biondi RM.

BiochimBiophysActa. 2013 Jul;1834(7):1302-21. doi: 10.1016/j.bbapap.2013.03.010. Epub 2013 Mar 21. Review.

PMID:

23524293

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Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

Chan TO, Zhang J, Rodeck U, Pascal JM, Armen RS, Spring M, Dumitru CD, Myers V, Li X, Cheung JY, Feldman AM.

Proc Natl AcadSci U S A. 2011 Nov 15;108(46):E1120-7. doi: 10.1073/pnas.1109879108. Epub 2011 Oct 26. PMID: 22031698

The nuts and bolts of AGC protein kinases. Pearce LR, Komander D, Alessi DR. Nat Rev Mol Cell Biol. 2010 Jan;11(1):9-22. doi: 10.1038/nrm2822. Review.

PMID: 20027184

Somatic mutations affect key pathways in lung adenocarcinoma.

Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, Sougnez C, Greulich H, Muzny DM, Morgan MB, Fulton L, Fulton RS, Zhang Q, Wendl MC, Lawrence MS, Larson DE, Chen K, Dooling DJ, Sabo A, Hawes AC, Shen H, Jhangiani SN, Lewis LR, Hall O, Zhu Y, Mathew T, Ren Y, Yao J, Scherer SE, Clerc K, Metcalf GA, Ng B, Milosavljevic A, Gonzalez-Garay ML, Osborne JR, Meyer R, Shi X, Tang Y, Koboldt DC, Lin L, Abbott R, Miner TL, Pohl C, Fewell G, Haipek C, Schmidt H, Dunford-Shore BH, Kraja A, Crosby SD, Sawyer CS, Vickery T, Sander S, Robinson J, Winckler W, Baldwin J, Chirieac LR, Dutt A, Fennell T, Hanna M, Johnson BE, Onofrio RC, Thomas RK, Tonon G, Weir BA, Zhao X, Ziaugra L, Zody MC, Giordano T, Orringer MB, Roth JA, Spitz MR, Wistuba II, Ozenberger B, Good PJ, Chang AC, Beer DG, Watson MA, Ladanyi M, Broderick S, Yoshizawa A, Travis WD, Pao W, Province MA, Weinstock GM, Varmus HE, Gabriel SB, Lander ES, Gibbs RA, Meyerson M, Wilson RK.

Nature. 2008 Oct 23;455(7216):1069-75. doi: 10.1038/nature07423. PMID: 18948947

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation. Hauge C, Antal TL, Hirschberg D, Doehn U, Thorup K, Idrissova L, Hansen K, Jensen ON, JÃ¸rgensen TJ, Biondi RM, FrÃ¶din M. EMBO J. 2007 May 2;26(9):2251-61. Epub 2007 Apr 19. PMID: 17446865

Structure, regulation and function of PKB/AKT--a major therapeutic target.

Hanada M, Feng J, Hemmings BA.

BiochimBiophysActa. 2004 Mar 11;1697(1-2):3-16. Review.

PMID: 15023346

AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation.

Chan TO, Rittenhouse SE, Tsichlis PN Annu Rev Biochem. 1999; 68():965-1014.

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MCB 2050 Lecture Notes - Lecture 5: Globulin, Protein Tag, Northern Blot

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