This document summarizes experiments purifying and characterizing a fusion protein of HdeA and its substrate Im7. It found that purification at 4°C with glycerol improved yields. His-tagged variants were screened for expression and purified, finding an N-terminal tag expressed proteins best. NMR analysis of a 15N-labeled fusion found signals, but degradation products complicated analysis. Mass spectrometry was used to identify degradation fragments to aid structural studies. Overall, the document outlines efforts to optimize a HdeA-Im7 fusion to study their interaction by NMR spectroscopy.
Graffinity: Novel affinity ligands for downstream processing of proteinsFrank Moffatt
Graffinity is a potentially revolutionary technology for the discovery of novel media for the purification of protein biologics. Proof of concept is sound. One example is a ligand that works as well as & better than protein A. We are open to suggestions or proposals for collaboration.
CONTACT fm@novalix-pharma.com
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
(originally aired 11-14-12)
Although biofuels are an attractive alternative to fossil fuels, large scale development is currently challenging. Development of renewable fuel characterization, processes, and contaminant analysis using robust analytical methods is needed. Here, focus is on Ion Chromatography—a proven technique for providing fast, reliable answers during research to production—with HPAE-PAD technology for carbohydrate analysis in feedstock and method parameter optimization (including column chemistry) for efficient separation of mono- and disaccharides with good resolution, linearity, and accuracy over a broad dynamic range. Since some residual sucrose and cellobiose may be present, examples of monitoring them and other saccharides is covered, along with their impact on the fermentation process.
Graffinity: Novel affinity ligands for downstream processing of proteinsFrank Moffatt
Graffinity is a potentially revolutionary technology for the discovery of novel media for the purification of protein biologics. Proof of concept is sound. One example is a ligand that works as well as & better than protein A. We are open to suggestions or proposals for collaboration.
CONTACT fm@novalix-pharma.com
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
(originally aired 11-14-12)
Although biofuels are an attractive alternative to fossil fuels, large scale development is currently challenging. Development of renewable fuel characterization, processes, and contaminant analysis using robust analytical methods is needed. Here, focus is on Ion Chromatography—a proven technique for providing fast, reliable answers during research to production—with HPAE-PAD technology for carbohydrate analysis in feedstock and method parameter optimization (including column chemistry) for efficient separation of mono- and disaccharides with good resolution, linearity, and accuracy over a broad dynamic range. Since some residual sucrose and cellobiose may be present, examples of monitoring them and other saccharides is covered, along with their impact on the fermentation process.
2024 State of Marketing Report – by HubspotMarius Sescu
https://www.hubspot.com/state-of-marketing
· Scaling relationships and proving ROI
· Social media is the place for search, sales, and service
· Authentic influencer partnerships fuel brand growth
· The strongest connections happen via call, click, chat, and camera.
· Time saved with AI leads to more creative work
· Seeking: A single source of truth
· TLDR; Get on social, try AI, and align your systems.
· More human marketing, powered by robots
ChatGPT is a revolutionary addition to the world since its introduction in 2022. A big shift in the sector of information gathering and processing happened because of this chatbot. What is the story of ChatGPT? How is the bot responding to prompts and generating contents? Swipe through these slides prepared by Expeed Software, a web development company regarding the development and technical intricacies of ChatGPT!
Product Design Trends in 2024 | Teenage EngineeringsPixeldarts
The realm of product design is a constantly changing environment where technology and style intersect. Every year introduces fresh challenges and exciting trends that mold the future of this captivating art form. In this piece, we delve into the significant trends set to influence the look and functionality of product design in the year 2024.
How Race, Age and Gender Shape Attitudes Towards Mental HealthThinkNow
Mental health has been in the news quite a bit lately. Dozens of U.S. states are currently suing Meta for contributing to the youth mental health crisis by inserting addictive features into their products, while the U.S. Surgeon General is touring the nation to bring awareness to the growing epidemic of loneliness and isolation. The country has endured periods of low national morale, such as in the 1970s when high inflation and the energy crisis worsened public sentiment following the Vietnam War. The current mood, however, feels different. Gallup recently reported that national mental health is at an all-time low, with few bright spots to lift spirits.
To better understand how Americans are feeling and their attitudes towards mental health in general, ThinkNow conducted a nationally representative quantitative survey of 1,500 respondents and found some interesting differences among ethnic, age and gender groups.
Technology
For example, 52% agree that technology and social media have a negative impact on mental health, but when broken out by race, 61% of Whites felt technology had a negative effect, and only 48% of Hispanics thought it did.
While technology has helped us keep in touch with friends and family in faraway places, it appears to have degraded our ability to connect in person. Staying connected online is a double-edged sword since the same news feed that brings us pictures of the grandkids and fluffy kittens also feeds us news about the wars in Israel and Ukraine, the dysfunction in Washington, the latest mass shooting and the climate crisis.
Hispanics may have a built-in defense against the isolation technology breeds, owing to their large, multigenerational households, strong social support systems, and tendency to use social media to stay connected with relatives abroad.
Age and Gender
When asked how individuals rate their mental health, men rate it higher than women by 11 percentage points, and Baby Boomers rank it highest at 83%, saying it’s good or excellent vs. 57% of Gen Z saying the same.
Gen Z spends the most amount of time on social media, so the notion that social media negatively affects mental health appears to be correlated. Unfortunately, Gen Z is also the generation that’s least comfortable discussing mental health concerns with healthcare professionals. Only 40% of them state they’re comfortable discussing their issues with a professional compared to 60% of Millennials and 65% of Boomers.
Race Affects Attitudes
As seen in previous research conducted by ThinkNow, Asian Americans lag other groups when it comes to awareness of mental health issues. Twenty-four percent of Asian Americans believe that having a mental health issue is a sign of weakness compared to the 16% average for all groups. Asians are also considerably less likely to be aware of mental health services in their communities (42% vs. 55%) and most likely to seek out information on social media (51% vs. 35%).
AI Trends in Creative Operations 2024 by Artwork Flow.pdfmarketingartwork
This article is all about what AI trends will emerge in the field of creative operations in 2024. All the marketers and brand builders should be aware of these trends for their further use and save themselves some time!
A report by thenetworkone and Kurio.
The contributing experts and agencies are (in an alphabetical order): Sylwia Rytel, Social Media Supervisor, 180heartbeats + JUNG v MATT (PL), Sharlene Jenner, Vice President - Director of Engagement Strategy, Abelson Taylor (USA), Alex Casanovas, Digital Director, Atrevia (ES), Dora Beilin, Senior Social Strategist, Barrett Hoffher (USA), Min Seo, Campaign Director, Brand New Agency (KR), Deshé M. Gully, Associate Strategist, Day One Agency (USA), Francesca Trevisan, Strategist, Different (IT), Trevor Crossman, CX and Digital Transformation Director; Olivia Hussey, Strategic Planner; Simi Srinarula, Social Media Manager, The Hallway (AUS), James Hebbert, Managing Director, Hylink (CN / UK), Mundy Álvarez, Planning Director; Pedro Rojas, Social Media Manager; Pancho González, CCO, Inbrax (CH), Oana Oprea, Head of Digital Planning, Jam Session Agency (RO), Amy Bottrill, Social Account Director, Launch (UK), Gaby Arriaga, Founder, Leonardo1452 (MX), Shantesh S Row, Creative Director, Liwa (UAE), Rajesh Mehta, Chief Strategy Officer; Dhruv Gaur, Digital Planning Lead; Leonie Mergulhao, Account Supervisor - Social Media & PR, Medulla (IN), Aurelija Plioplytė, Head of Digital & Social, Not Perfect (LI), Daiana Khaidargaliyeva, Account Manager, Osaka Labs (UK / USA), Stefanie Söhnchen, Vice President Digital, PIABO Communications (DE), Elisabeth Winiartati, Managing Consultant, Head of Global Integrated Communications; Lydia Aprina, Account Manager, Integrated Marketing and Communications; Nita Prabowo, Account Manager, Integrated Marketing and Communications; Okhi, Web Developer, PNTR Group (ID), Kei Obusan, Insights Director; Daffi Ranandi, Insights Manager, Radarr (SG), Gautam Reghunath, Co-founder & CEO, Talented (IN), Donagh Humphreys, Head of Social and Digital Innovation, THINKHOUSE (IRE), Sarah Yim, Strategy Director, Zulu Alpha Kilo (CA).
Trends In Paid Search: Navigating The Digital Landscape In 2024Search Engine Journal
The search marketing landscape is evolving rapidly with new technologies, and professionals, like you, rely on innovative paid search strategies to meet changing demands.
It’s important that you’re ready to implement new strategies in 2024.
Check this out and learn the top trends in paid search advertising that are expected to gain traction, so you can drive higher ROI more efficiently in 2024.
You’ll learn:
- The latest trends in AI and automation, and what this means for an evolving paid search ecosystem.
- New developments in privacy and data regulation.
- Emerging ad formats that are expected to make an impact next year.
Watch Sreekant Lanka from iQuanti and Irina Klein from OneMain Financial as they dive into the future of paid search and explore the trends, strategies, and technologies that will shape the search marketing landscape.
If you’re looking to assess your paid search strategy and design an industry-aligned plan for 2024, then this webinar is for you.
5 Public speaking tips from TED - Visualized summarySpeakerHub
From their humble beginnings in 1984, TED has grown into the world’s most powerful amplifier for speakers and thought-leaders to share their ideas. They have over 2,400 filmed talks (not including the 30,000+ TEDx videos) freely available online, and have hosted over 17,500 events around the world.
With over one billion views in a year, it’s no wonder that so many speakers are looking to TED for ideas on how to share their message more effectively.
The article “5 Public-Speaking Tips TED Gives Its Speakers”, by Carmine Gallo for Forbes, gives speakers five practical ways to connect with their audience, and effectively share their ideas on stage.
Whether you are gearing up to get on a TED stage yourself, or just want to master the skills that so many of their speakers possess, these tips and quotes from Chris Anderson, the TED Talks Curator, will encourage you to make the most impactful impression on your audience.
See the full article and more summaries like this on SpeakerHub here: https://speakerhub.com/blog/5-presentation-tips-ted-gives-its-speakers
See the original article on Forbes here:
http://www.forbes.com/forbes/welcome/?toURL=http://www.forbes.com/sites/carminegallo/2016/05/06/5-public-speaking-tips-ted-gives-its-speakers/&refURL=&referrer=#5c07a8221d9b
ChatGPT and the Future of Work - Clark Boyd Clark Boyd
Everyone is in agreement that ChatGPT (and other generative AI tools) will shape the future of work. Yet there is little consensus on exactly how, when, and to what extent this technology will change our world.
Businesses that extract maximum value from ChatGPT will use it as a collaborative tool for everything from brainstorming to technical maintenance.
For individuals, now is the time to pinpoint the skills the future professional will need to thrive in the AI age.
Check out this presentation to understand what ChatGPT is, how it will shape the future of work, and how you can prepare to take advantage.
A brief introduction to DataScience with explaining of the concepts, algorithms, machine learning, supervised and unsupervised learning, clustering, statistics, data preprocessing, real-world applications etc.
It's part of a Data Science Corner Campaign where I will be discussing the fundamentals of DataScience, AIML, Statistics etc.
Time Management & Productivity - Best PracticesVit Horky
Here's my presentation on by proven best practices how to manage your work time effectively and how to improve your productivity. It includes practical tips and how to use tools such as Slack, Google Apps, Hubspot, Google Calendar, Gmail and others.
The six step guide to practical project managementMindGenius
The six step guide to practical project management
If you think managing projects is too difficult, think again.
We’ve stripped back project management processes to the
basics – to make it quicker and easier, without sacrificing
the vital ingredients for success.
“If you’re looking for some real-world guidance, then The Six Step Guide to Practical Project Management will help.”
Dr Andrew Makar, Tactical Project Management
2024 State of Marketing Report – by HubspotMarius Sescu
https://www.hubspot.com/state-of-marketing
· Scaling relationships and proving ROI
· Social media is the place for search, sales, and service
· Authentic influencer partnerships fuel brand growth
· The strongest connections happen via call, click, chat, and camera.
· Time saved with AI leads to more creative work
· Seeking: A single source of truth
· TLDR; Get on social, try AI, and align your systems.
· More human marketing, powered by robots
ChatGPT is a revolutionary addition to the world since its introduction in 2022. A big shift in the sector of information gathering and processing happened because of this chatbot. What is the story of ChatGPT? How is the bot responding to prompts and generating contents? Swipe through these slides prepared by Expeed Software, a web development company regarding the development and technical intricacies of ChatGPT!
Product Design Trends in 2024 | Teenage EngineeringsPixeldarts
The realm of product design is a constantly changing environment where technology and style intersect. Every year introduces fresh challenges and exciting trends that mold the future of this captivating art form. In this piece, we delve into the significant trends set to influence the look and functionality of product design in the year 2024.
How Race, Age and Gender Shape Attitudes Towards Mental HealthThinkNow
Mental health has been in the news quite a bit lately. Dozens of U.S. states are currently suing Meta for contributing to the youth mental health crisis by inserting addictive features into their products, while the U.S. Surgeon General is touring the nation to bring awareness to the growing epidemic of loneliness and isolation. The country has endured periods of low national morale, such as in the 1970s when high inflation and the energy crisis worsened public sentiment following the Vietnam War. The current mood, however, feels different. Gallup recently reported that national mental health is at an all-time low, with few bright spots to lift spirits.
To better understand how Americans are feeling and their attitudes towards mental health in general, ThinkNow conducted a nationally representative quantitative survey of 1,500 respondents and found some interesting differences among ethnic, age and gender groups.
Technology
For example, 52% agree that technology and social media have a negative impact on mental health, but when broken out by race, 61% of Whites felt technology had a negative effect, and only 48% of Hispanics thought it did.
While technology has helped us keep in touch with friends and family in faraway places, it appears to have degraded our ability to connect in person. Staying connected online is a double-edged sword since the same news feed that brings us pictures of the grandkids and fluffy kittens also feeds us news about the wars in Israel and Ukraine, the dysfunction in Washington, the latest mass shooting and the climate crisis.
Hispanics may have a built-in defense against the isolation technology breeds, owing to their large, multigenerational households, strong social support systems, and tendency to use social media to stay connected with relatives abroad.
Age and Gender
When asked how individuals rate their mental health, men rate it higher than women by 11 percentage points, and Baby Boomers rank it highest at 83%, saying it’s good or excellent vs. 57% of Gen Z saying the same.
Gen Z spends the most amount of time on social media, so the notion that social media negatively affects mental health appears to be correlated. Unfortunately, Gen Z is also the generation that’s least comfortable discussing mental health concerns with healthcare professionals. Only 40% of them state they’re comfortable discussing their issues with a professional compared to 60% of Millennials and 65% of Boomers.
Race Affects Attitudes
As seen in previous research conducted by ThinkNow, Asian Americans lag other groups when it comes to awareness of mental health issues. Twenty-four percent of Asian Americans believe that having a mental health issue is a sign of weakness compared to the 16% average for all groups. Asians are also considerably less likely to be aware of mental health services in their communities (42% vs. 55%) and most likely to seek out information on social media (51% vs. 35%).
AI Trends in Creative Operations 2024 by Artwork Flow.pdfmarketingartwork
This article is all about what AI trends will emerge in the field of creative operations in 2024. All the marketers and brand builders should be aware of these trends for their further use and save themselves some time!
A report by thenetworkone and Kurio.
The contributing experts and agencies are (in an alphabetical order): Sylwia Rytel, Social Media Supervisor, 180heartbeats + JUNG v MATT (PL), Sharlene Jenner, Vice President - Director of Engagement Strategy, Abelson Taylor (USA), Alex Casanovas, Digital Director, Atrevia (ES), Dora Beilin, Senior Social Strategist, Barrett Hoffher (USA), Min Seo, Campaign Director, Brand New Agency (KR), Deshé M. Gully, Associate Strategist, Day One Agency (USA), Francesca Trevisan, Strategist, Different (IT), Trevor Crossman, CX and Digital Transformation Director; Olivia Hussey, Strategic Planner; Simi Srinarula, Social Media Manager, The Hallway (AUS), James Hebbert, Managing Director, Hylink (CN / UK), Mundy Álvarez, Planning Director; Pedro Rojas, Social Media Manager; Pancho González, CCO, Inbrax (CH), Oana Oprea, Head of Digital Planning, Jam Session Agency (RO), Amy Bottrill, Social Account Director, Launch (UK), Gaby Arriaga, Founder, Leonardo1452 (MX), Shantesh S Row, Creative Director, Liwa (UAE), Rajesh Mehta, Chief Strategy Officer; Dhruv Gaur, Digital Planning Lead; Leonie Mergulhao, Account Supervisor - Social Media & PR, Medulla (IN), Aurelija Plioplytė, Head of Digital & Social, Not Perfect (LI), Daiana Khaidargaliyeva, Account Manager, Osaka Labs (UK / USA), Stefanie Söhnchen, Vice President Digital, PIABO Communications (DE), Elisabeth Winiartati, Managing Consultant, Head of Global Integrated Communications; Lydia Aprina, Account Manager, Integrated Marketing and Communications; Nita Prabowo, Account Manager, Integrated Marketing and Communications; Okhi, Web Developer, PNTR Group (ID), Kei Obusan, Insights Director; Daffi Ranandi, Insights Manager, Radarr (SG), Gautam Reghunath, Co-founder & CEO, Talented (IN), Donagh Humphreys, Head of Social and Digital Innovation, THINKHOUSE (IRE), Sarah Yim, Strategy Director, Zulu Alpha Kilo (CA).
Trends In Paid Search: Navigating The Digital Landscape In 2024Search Engine Journal
The search marketing landscape is evolving rapidly with new technologies, and professionals, like you, rely on innovative paid search strategies to meet changing demands.
It’s important that you’re ready to implement new strategies in 2024.
Check this out and learn the top trends in paid search advertising that are expected to gain traction, so you can drive higher ROI more efficiently in 2024.
You’ll learn:
- The latest trends in AI and automation, and what this means for an evolving paid search ecosystem.
- New developments in privacy and data regulation.
- Emerging ad formats that are expected to make an impact next year.
Watch Sreekant Lanka from iQuanti and Irina Klein from OneMain Financial as they dive into the future of paid search and explore the trends, strategies, and technologies that will shape the search marketing landscape.
If you’re looking to assess your paid search strategy and design an industry-aligned plan for 2024, then this webinar is for you.
5 Public speaking tips from TED - Visualized summarySpeakerHub
From their humble beginnings in 1984, TED has grown into the world’s most powerful amplifier for speakers and thought-leaders to share their ideas. They have over 2,400 filmed talks (not including the 30,000+ TEDx videos) freely available online, and have hosted over 17,500 events around the world.
With over one billion views in a year, it’s no wonder that so many speakers are looking to TED for ideas on how to share their message more effectively.
The article “5 Public-Speaking Tips TED Gives Its Speakers”, by Carmine Gallo for Forbes, gives speakers five practical ways to connect with their audience, and effectively share their ideas on stage.
Whether you are gearing up to get on a TED stage yourself, or just want to master the skills that so many of their speakers possess, these tips and quotes from Chris Anderson, the TED Talks Curator, will encourage you to make the most impactful impression on your audience.
See the full article and more summaries like this on SpeakerHub here: https://speakerhub.com/blog/5-presentation-tips-ted-gives-its-speakers
See the original article on Forbes here:
http://www.forbes.com/forbes/welcome/?toURL=http://www.forbes.com/sites/carminegallo/2016/05/06/5-public-speaking-tips-ted-gives-its-speakers/&refURL=&referrer=#5c07a8221d9b
ChatGPT and the Future of Work - Clark Boyd Clark Boyd
Everyone is in agreement that ChatGPT (and other generative AI tools) will shape the future of work. Yet there is little consensus on exactly how, when, and to what extent this technology will change our world.
Businesses that extract maximum value from ChatGPT will use it as a collaborative tool for everything from brainstorming to technical maintenance.
For individuals, now is the time to pinpoint the skills the future professional will need to thrive in the AI age.
Check out this presentation to understand what ChatGPT is, how it will shape the future of work, and how you can prepare to take advantage.
A brief introduction to DataScience with explaining of the concepts, algorithms, machine learning, supervised and unsupervised learning, clustering, statistics, data preprocessing, real-world applications etc.
It's part of a Data Science Corner Campaign where I will be discussing the fundamentals of DataScience, AIML, Statistics etc.
Time Management & Productivity - Best PracticesVit Horky
Here's my presentation on by proven best practices how to manage your work time effectively and how to improve your productivity. It includes practical tips and how to use tools such as Slack, Google Apps, Hubspot, Google Calendar, Gmail and others.
The six step guide to practical project managementMindGenius
The six step guide to practical project management
If you think managing projects is too difficult, think again.
We’ve stripped back project management processes to the
basics – to make it quicker and easier, without sacrificing
the vital ingredients for success.
“If you’re looking for some real-world guidance, then The Six Step Guide to Practical Project Management will help.”
Dr Andrew Makar, Tactical Project Management
4. Problem that previously occurred
during purification of HdeA D20A
D51A from minimal medium
load
25
20
15
10
25
20
15
10
Increasing NaCl concentration during elution from HiTrap SP HP
HdeA
bla
5. PurificationsetupRT vs 4C
Cell Culture
Periplasmic
protein
extraction
Dialysis
Purification at
Room Temp/No
Glycerol
Purification at
4o C/ 5%
Glycerol
15. Future Directions Part I
Purification of the constitutively active
mutant HdeA D20A D51A
• Purify further with a Phenyl Column to attempt to remove
remaining contaminating proteins
• Redo the entire protocol using bacteria grown in 15N Labeled
media.
• Purify that protein and use it in NMR analysis
20. Variable 2: Variations of Im7
I22V
L18A
L19A
L37A
I54A
L53A
Largely destabilized Partially unfolded
Model for intermediate state
Model for unfolded state
21. Variable 3:
N- or C- Terminal 6xHis-tag
ss-HdeA – (GGGGS)2 – Im7 – GSG – 6xHis
Im7Linker
ss-6xHis-GSG-HdeA – (GGGGS)2 – Im7
HdeA
Im7Linker
His Tag
His Tag
HdeASS
SS
22. His Tag Rationale
• Protein was not completely pure after native
purification
• Still contaminating bands, probably degradation
products
23. His Tag Rationale
• Previous experiments demonstrated significant contamination
of the periplasmic extracts with potential degradation
products in addition to the fusion protein of interest
HdeA F35W-(GGGGS)2-Im7 L53A I54A ∆W
(LF 1434)
*
Full length
protein
Degradation
products?
*
*
20 ul
load
10 ul
load
24. His Tag Rationale
• Previous experiments demonstrated significant contamination
of the periplasmic extracts with potential degradation
products in addition to the fusion protein of interest
• Adding a His-tag creates a system designed to quickly screen
multiple types of proteins using one purification technique for
their suitability in NMR analysis
25. Screening for expression of the His-
tagged fusion proteins
1. Is the his-tagged fusion proteins expressed at all?
2. Is an N- or a C-terminal tag preferred?
• expression
• purification
27. Purification of His-Tagged HdeA-
Im7 Fusion Protein LF 1508
Im7Linker
His Tag
HdeASS
1508: Wild Type I22V His Tag
C Terminal His Tag
28. Increasing Concentration of Imidazole
35
25
15
kDa
35
25
15
kDa
ElutionFractionsof LF1508Purification
Im7Linker
His Tag
HdeASS
1508: Wild Type I22V His Tag
C Terminal
Fusion
Protein
*Pure Products
29. 1. NMR attemptwith a 15N HdeA-Im7 fusion
(HdeA WT-(GGGGS)2-Im7I22V-GSG-6xHis)
HiTrap Chelating
15N minimal medium
Purification Ke Wan
30. HSQC spectrum of
HdeA WT-(GGGGS)2-Im7 I22V-GSG-
6xHis
1H
15N
Expected # of peaks: 185
Buffer
50 mM KH2PO4
90 mM NaCl
1 mM DSS
0.5 mM EDTA
1 mM chloroacetic acid
5% D2O
pH 2.5
Scott
33. HSQC spectra 15N Im7 I22V +
HdeA
1H [ppm]
15N[ppm]
500 uM 15N Im7 I22V
500 uM 15N Im7 I22V + 571 uM HdeA
Linda + Loic
34. Im7 L18A L37A L38A might be a
better substrate for NMR
90% 1H2O
10% 2H2O
0.2 M
Na2SO4
10C
Pashley, C. L. et al. (2011): Journal of Molecular Biology (2011)
35. Purification of His-Tagged HdeA-
Im7 Fusion Protein LF 1510
Im7
HdeA Linker
His TagSS
N Terminal His Tag
1510: Wild Type L18A L19A L37AHis Tag
36. Elution Fractions of LF1510 Purification
Im7HdeA Linker
His TagSS
N Terminal His Tag
1510: Wild Type L18A L19A L37AHis Tag
Load B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6 C7 C8
25
20
37
Kda
37. Purification of His-Tagged HdeA-
Im7 Fusion Protein LF 1507
Im7Linker
His Tag
HdeASS
1507: Wild Type L18A L19A L37A His Tag
C Terminal His Tag
38. Im7Linker
His Tag
HdeASS
1507: Wild Type L18A L19A L37A His Tag
C Terminal His Tag
Elution Fractions of LF1507 Purification
Load FT Wash A8 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7
25
20
37
Kda
42. Degradation products (?) also
occur after purification I
HdeA F35W-(GGGGS)2-Im7 L53A I54A ∆W
(LF 1434)
*
Full length
protein
Degradation
products?
*
*
20 ul
load
10 ul
load
LINDA
43. Degradation products (?) also
occur after purification II
Selected elution fractions purification
HdeA WT-(GGGGS)2-
Im7 L18A L19A L37A-GSG-6xHis
(LF1507)
20
15
25
10
*
*
*
Full length
protein
Degradation
Product(s)?
20
15
25
10
Selected elution fractions purification
HdeA WT-(GGGGS)2-
Im7 I22V-GSG-6xHis
(LF1508)
*
*
44. C-terminal His-tag cannot be
detected for degradation products I
Selected elution fractions purification
HdeA WT-(GGGGS)2-
Im7 L18A L19A L37A-GSG-6xHis
(LF1507)
20
15
25
10
*
*
*
Anti 6xHis antibody
20
15
25
10
Full length
protein
Degradation
Product(s)?
*
47. N-terminal His-tag is present
however
Im7
HdeA Linker
His TagSS
N Terminal His Tag
1510: Wild Type L18A L19A L37AHis Tag
Elution fractions HiTrap Chelating column
48. Identify degradation products by mass
spectrometry (UofM Bioconsortium)
HdeA F35W-(GGGGS)2-
Im7 L53A I54A ∆W
(LF 1434)
*
Full length
protein
Degradation
products?
*
*
20 ul
load
10 ul
load
Cut out bands Proteolytic digest Identification of peptides
His-tag-GSG-HdeA WT-(GGGGS)2-
Im7 L18A L19A L37A
(LF1510)
49. Identify degradation products by mass
spectrometry (Indiana University)
LF 1434 HdeA F35W-link-Im7 L53A I54A W75F
LF 1508 HdeA WT -link-Im7 I22V-6xHis
LF 1510 6xHis-HdeA WT -link-Im7 L18A L19A L37A
Cut out bands Proteolytic digest
identification of peptides
Intact mass determination
50. Mass spec data turnaround
University of Michigan
Proteomics Core
Indiana University
52. Future experiments
• 15N labeling of HdeA D20A D51A
structural studies with NMR
• Identification of degradation products of the HdeA-Im7
fusions
use stable degradation products for structural studies
instead of full length protein
• Create fusion proteins between HdeA and peptides previously
identified as potential HdeA substrates in peptide array
Protein will not bind to anion exchange when employed as a first step
HdeA D20A D51A aggregates on the cation exchange column when purified from minimal medium
Problem does not occur when protein is purified from rich medium
Hypothesis: the protein is binding to the column
Hypothesis: the protein is binding to the column
This is showing that the first large UV vis peak is due to elution of mostly beta lactamase, and to show that the TINY amount of HdeA const. mutant we’re getting is coming in fractions after the end of the peak
Hypothesis: the protein is binding to the column
Hypothesis: the protein is binding to the column
Again showing at 4 degree C/5% glycerol the beta lactamase comes off mostly in the first peak
This now shows that the decrease in the first peak is because a lot of beta lactamase has already come off – yes it’s still there all the way through, but now the second peak is also do to a large amount of HdeA coming off
Linker artificially should create a 1:1 ratio of HdeA and Im7 to allow us to better study the interactions between these two proteins without the problem described earlier of precipitation of Im7
Different linker lengths still allow us to maintain a constant HdeA: Im7 ratio, but allow us to additionally study how distance might affect –
Kinetics of the interaction of the proteins
Also if the linker might be too short, and affect the ability of the proteins to interact at all
This will allow us to test the thermodynamics of the interactions between Im7 and HdeA
With the previous variable we’ll be able to determine how quickly it takes for Im7 and HdeA to bind
This variable will allow us to determine what mutations might weaken the intermolecular forces between the two proteins
Correction: we did the expression test to see
1 if the his-tagged fusion proteins would be expressed at all- to do this we collected proteins from bacteria transformed to express various constructs. The parameters were HdeA WT (variable a = linker length) (variable b = Im7 variant) (variable c = linker attaches Im7 to HdeA either on its
2 is N- or C-terminal tagged proteins had different or the same levels of expression
We used 4 different mutants of Im7
Show Im7 structure with mutations indicated here instead of table
Label different lanes, indicate which band we are interested in
Do we still have the samples and could run this gel again (longer?)
Can be purified to high purity
Show NMR spectrum here, Linda will provide the slide
HdeA gives a lot of info to us when we perform NMR spectra on it
Fig. 7. The 500-MHz 1H–15N HSQC spectrum of L18A–L19A–L37A. The spectrum was acquired in buffer A (90% 1H2O/10% 2H2O) with 0.2 M Na2SO4 at 10 °C. Sequential NOEs are observed for NH groups of i and i±1 residues for stretches of the protein involving residues 8–17, 18–37, 41–45, 51–52, 54–55, 60–63 and 67–69; however, no longer-range NOEs were observed in the three-dimensional HSQC-NOESY-HSQC experiment. The expanded region shows the assignments in crowded regions more clearly.
Since that variant (triple alanine) gave such a better spectra, we decided to try to purify the mutant created by Linda that expressed Im7 variant 3A in the fusion protein.