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Training Program on Protein Expression, Purification, and Analysis of Biomolecules

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To encourage more Stanford researchers to engage in structural biology and train them to better produce, purify, and characterize proteins, the Macromolecular Structure Knowledge Center (MSKC) at Sarafan ChEM-H is accepting proposals from Stanford scientists who would like to start or enhance a protein characterization project in their lab. Selected projects will receive personalized, hands-on training in a state-of-the-art facility and up to $7,500 in matching funds towards the project.

Deadline:

To apply, submit proposals through the SlideRoom application portal below by 11:59 p.m. on Friday, February 24, 2023.

Apply Here

Eligibility: 

Each application must identify one Principal Investigator (Stanford faculty with UTL, UML, NTLR and CE faculty appointments; Instructors; and Clinical Instructors are all eligible) and one Primary Researcher (an undergraduate student, graduate student or postdoctoral scholar, clinical or non-clinical, in a Stanford lab). PI waivers are not required for this program.

Overview and Program Goals:

The objective of the MSKC Training Program is to encourage the exploration of new protein projects by working directly with researchers on protein production, purification, and biophysical analysis, and up to three-dimensional structure methodologies. The aim is for training personnel coming from different fields of research, regardless of background, to generate pilot data, in support of three-dimensional structure-related research, in early phase investigations, and preliminary data in support of research proposals for external funding agencies.  MSKC makes tools available to awardees to advance protein projects from ideas to reality, from a sequence on paper to a high-quality sample for any downstream application. Each proposal should highlight the transformative potential the protein project would have on your research. Additionally, projects in support of existing three-dimensional structure-related research areas, early phase investigations, and preliminary data for extramural proposals will be given priority. Basic research directed towards protein characterization is highly encouraged.

Support Provided:

Support will be provided in the form of one-on-one training and access to instrumentation at each stage of the protein production, purification, crystallization, and characterization, including:

  • Hands-on protein production using the E. coli recombinant expression system and purification supported by chromatographic methods via Akta Pure Fast Protein Liquid Chromatography (FPLC).
  • Biophysical analysis, protein structure stability and oligomerization, and ligand binding affinity measurements through NanoTemper Panta and Monolith Labelfree instruments.
  • Advanced three-dimensional structure methodologies supported by Douglas Instruments, Formulatrix, and synchrotron macromolecular crystallography (MX) via SSRL/SLAC. 

In addition, matching funds of up to $7,500 will be provided.

Requirements:

Each application submission will include the following application materials:

  1. Project Summary (200 words) 
    • A non-confidential, 200-word summary of the project, focusing on the motivation and goals of the project. This should be written  with a lay audience in mind. This may be used on the Sarafan ChEM-H website or in other public-facing documents.
  2. Research Proposal 
    • Details below
  3. Letter of acknowledgement
    • The application must include a signed letter from the Principal Investigator acknowledging that:
      • They will financially support the project to at least match, and if necessary exceed, any funding provided through this opportunity. 
      • They will support the designated Primary Researcher in working in the MSKC for the duration of the project. 
      • They will supply any consumables or other supplies not readily stocked in the MSKC.

You do not need to submit your applications to your Research Process Manager (RPM) in RMG or through your Office of Sponsored Research (OSR) Contract and Grant officer (CGO) for their approval at this time.

Note: We will require a final report at the conclusion of the project and request you to inform us of any publications, patents, presentations, and additional funding that result from this program in the future.

Research Proposal Structure:

Proposals should be organized as follows:

  1. Title Page (1 page)
    • Project Title
    • Name, title, and contact information for Principal Investigator and  Primary Researcher
  2. Proposal (2 pages)
    • Project narrative and figures, if relevant, to explain the background and aims of the project. Proposals should be concise and practical. Include the following sections:
      • Project background 
      • Project goals (describe the desired outcomes and potential scientific impact)
      • Training goals (describe role of MSKC training and the significance of MSKC technology on your project)

Selection:

Proposals will be reviewed by a faculty committee for scientific merit and then assessed by MSKC personnel for feasibility under the nature of the award funds and timeframe. Final decisions on full proposals will be made by late March, 2023.

About the MSKC:

The Macromolecular Structure Knowledge Center (MSKC) at Sarafan ChEM-H is a hybrid service center-research laboratory providing access to technologies and expertise to researchers carrying out biological experiments. The MSKC is located in the Sarafan ChEM-H/Wu Tsai Neurosciences Institute Building at the crossroads between the Schools of Engineering, Medicine, and Humanities and Sciences. The MSKC is a training ground to anyone - undergraduate and graduate students, postdoctoral scholars, and staff researchers - interested in protein work regardless of their scientific areas of expertise. The MSKC offers an experiential learning and training program aimed at teaching the fundamentals for producing, purifying, and structurally characterizing biological macromolecules. With this call, MSKC is particularly interested in training awardees in fundamental tools and technologies within protein biology to meet the evolving needs of researchers. 

Project Timelines and Budgets:

At the bottom of this page, you will find a detailed explanation of typical timelines and average hands-on time to complete projects in the MSKC. These projects will take at least one month to complete, but typically closer to three months.

Not all projects will follow the same timeline. There are typically two expected deliverables:

  1. Production of the molecular sample in large quantity and at high purity
  2. Identifying crystallization conditions to produce crystals of the sample

Projects, on average, will require at least $15,000 to complete ($7,500 provided by the research lab and $7,500 in matching funds), with approximately $10,000 needed for step (1) and $5,000 for step (2). As resolving crystal structures can be challenging, we cannot guarantee that all projects will result in a crystal structure.

Sample Information:

A single protein sequence (full-length under 90 kDa mass) will be considered. If more than one protein sample per project would be required, e.g. for a multi-protein complex, it should be sufficiently justified in the proposal. A UniProt Knowledgebase (UniProtKB) entry ID, or the amino acid sequence, and/or the protein name should be included in the proposal.  

Questions? Contact Daniel Fernandez, PhD, MSKC Director, at mskc-staff@stanford.edu

Training Structure:

Protein Expression

We use bacterial expression as a factory to obtain the protein at large quantities for downstream applications. Under the MSKC staff supervision, the primary researcher will be trained to safely work with the well established bacterial strain E. coli. In this module the primary researcher will acquire concepts and will work on: preparing the stage to work in aseptic conditions; assess bacterial growth in solid and in liquid media; prepare solutions (buffers, salts, agar); sterilize material by filtering and autoclaving; use of centrifuge to pellet cells; prepare Erlenmeyer flasks for cell growth in an orbital shaking system; use of sonicator to break down cells; separate cell debris from liquid supernatant; safely dispose of cell debris; appropriately preserve sample for downstream processing.

Protein Purification and Biophysical Analysis

This module is aimed to train the primary researcher in techniques to separate the protein of interest from contaminants and assess the degree of purity of the sample. The primary researcher will be introduced to the concepts of affinity chromatography and size exclusion using an AKTA Pure FPLC system. Sample purity will be determined by SDS-PAGE electrophoresis running on precast acrylamide gels and Coomassie blue staining. The protein sample concentration will be measured by absorbance on a nanodrop spectrophotometer. Using a centrifugal filter device, aliquoting and appropriately storing the sample. Protein stability, aggregation, and thermal unfolding will be determined by the Prometheus Panta system. Ligand-binding affinity measurements will be done with the Monolith Labelfree using known ligands of the protein and/or from screening potential new ligands from a compounds library.

Protein Crystallization 

This module introduces crystallization and allied techniques aimed at obtaining a detailed molecular picture of the protein of interest. The sample will be mixed with a series of pre-made solutions while deposited on a transparent, 96-well container (crystal tray) that is sealed and incubated at different temperatures. The evolution of the crystallization mix will be followed by eye inspection under an optical microscope and by an automated imaging Formulatrix imager on a weekly basis. Crystallization will then be used to introduce the primary researcher to X-ray diffraction experiments and 3D structure determination.

Work Timeline:

Illustrative time requirements of the different modules are indicated in the table below.

Module Length of time (estimate) Expected primary researcher commitment Time (h) in MSKC (estimate)
Protein expression (E. coli) 1 week

2 hr safety training/lab orientation

4 hr prepare material

2 hr prepare cell inoculum/culture

4 hr cell induction/growth follow up

4 hr pellet cells/sonicate/lysate prep

16
Protein purification and biophysical analysis 2 weeks

8 hr FPLC affinity + size exclusion purification

2 hr SDS-PAGE electrophoresis

6 hr stability analysis (Prometheus Panta)

6 hr binding affinity measurements (Monolith LabelFree)

2 hr prepare for crystallization

 

24
Protein crystallization 1 week*

4 hr set up crystal trays

2 hr image crystal trays/twice

8

*Tentative, but at least 1 week, and up to 4-6 weeks (crystals grow over long periods of time and further imaging will be needed).

Note: Timing required for crystallization is sample-dependent and quite variable.