HTP Lab

W. Clay Brown, Ph.D., Scientific Director
Room 3338D

About

The LSI Center for Structural Biology’s High-throughput Protein Lab can help you obtain the protein you need for your research.

With extensive expertise and experience with eukaryotic and prokaryotic proteins, we offer the evaluation of expression yield from multiple gene constructs, vectors, growth conditions and expression systems to determine which combinations can meet the needs of your project.

This increased “bandwidth” speeds you to answers to your protein questions.

Our processes cover high-throughput cloning and expression testing in:

  • bacterial systems (96-well blocks)

  • baculovirus-infected insect cell systems (24-well blocks, up to 48)

  • mammalian cell systems (also processed in 24-well blocks)

Processing Time

The average length of expression testing is:

  • 3-5 weeks for bacteria
  • 6-8 weeks for baculovirus
  • 3-5 weeks for mammalian cells

Vector Libraries

At the heart of our process lie the vector libraries that we have constructed.

Our vectors contain the ligation-independent cloning (LIC) region developed at the Midwest Center for Structural Genomics (MCSG). This allows for rapid and efficient cloning of large numbers of constructs, independent of restriction sites, in many vectors simultaneously.

And while our bacterial process began with the MCSG-developed vectors, we have expanded on these to incorporate a larger number of fusion partners as well as various promoters of different strengths. The fusion and promoter variants are all repeated in three series of bacterial vectors (>50 in all) that vary by replication origin and selective marker, which allows for members from different series to be used independently or together for co-expression experiments, depending on the needs of your project.

A series of baculovirus vectors has since developed employing the same LIC region as found in the bacterial vectors. The different fusions were combined with three different signal sequences (17 in all) to afford the greatest degree of flexibility in experimental design. A set of mammalian cytosolic and expression vectors based on pCDNA3.1 have also been constructed with the same LIC region and fusions and were then duplicated with a signal sequence (from Cox2) for secretion into the growth medium.

The use of the same LIC region in all of our vectors allows us to clone a single gene or fragment into many bacterial, baculovirus and mammalian expression vectors and carry out expression testing simultaneously.

Getting Started

Contact the HTP lab to discuss the goals of your project, what you want to do with your protein(s) and what you know about that protein.

It is very useful for us to know what you have tried or not tried and what your data look like. From this we can develop a strategy with you for providing the materials that will best meet your needs.

We do not do primary cloning and will require a starting clone to use as a PCR template. We generally request 1-2 microliters of miniprep quality DNA per construct that we have planned. This is sufficient for several rounds of PCR should that be required. We will also need the DNA sequence of your target for use in designing oligos for PCR.

Services

Small- & Large-Scale Projects

All the data and materials generated as well as protocols and recommendations for duplicating our work at larger scale are provided to our collaborators. Recognizing that some labs may not have the expertise or equipment to culture insect cells, we provide a scale-up service for this system.

If relatively small amounts of protein are required, cell paste may be generated using shake-flasks. If larger amounts of protein are needed, we can employ a bioreactor with a working volume of 20 L. As we develop our capabilities with mammalian cell expression evaluation we plan to provide similar scale-up service for this system as well.

Project Analysis & Design

Information provided by the requesting lab, such as known biochemistry and planned applications, is combined with bioinformatic analysis using a variety of server-based software.

This analysis evaluates predicted areas of folding and disorder, secondary structure prediction, areas of homology and biochemical activity motifs. The possible domain boundaries are identified and potential construct limits are proposed. The gene is also analyzed for codon usage in E. coli. A cloning strategy taking advantage of a substantial vector library is developed. The resulting plan is submitted to the requesting lab for review. Clonemanager software is then used to design and analyze oligos for PCR construction that are compatible with ligation-independent cloning.

Training

We familiarize users with server-based analysis software —Foldindex, DisEMBL, Globplot, Jalview and Jpred — and demonstrate how the tools are applied, how to use the information from the analysis and what to concentrate on for construct design.

Consulting

We consult on oligo design, load sequences of templates, and use of Clonemanager software to design, analyze and finalize sequences for ordering.

PCR Construction

Using a template provided by the requesting lab, the various gene constructs are produced by PCR. PCR products are purified in 96-well plate format on the Biomek and then analyzed using the Labchip 90.

Ligation-Independent Cloning

PCR fragments are prepared for LIC by treatment with T4 DNA polymerase. These are annealed into the appropriate vectors and used to transform XL1 Blue cells. Transformants are plated onto 48-well Q-trays and incubated overnight. Colonies are picked and used to inoculate 1 ml of media in each well of a 96-well block. These cultures are grown overnight and then plasmid DNA is purified by miniprepping in a 96-well plate on the Biomek. Positive clones are identified by PCR followed by Labchip analysis.

Training

We provide training on LIC protocols, guidance in our lab for PCR construction, processing for LIC, annealing, transformation and screening for positive clones, using the Biomek for minipreps and the Labchip for PCR analysis.

Baculovirus Services

Positive clones obtained are used to make recombinant baculovirus using the Bac to Bac system. Recombinant bacmids are purified using traditional alkali lysis miniprep protocols. The bacmids are screened by PCR using M13 primers to confirm insertion of the gene of interest into the bacmid. The bacmid DNAs are used to transfect insect cells in suspension in 24-well blocks. Transfection in suspension has been shown to yield higher viral titers. The resulting virus is titered by QPCR. Virus titering by QPCR can be requested as an individual service.

Mammalian services

Positive clones are identified in pCDNA3.1-based vectors that have been modified for LIC using the same cloning region found in our bacterial and baculovirus vectors. These provide a variety of fusion tags from His to FLAG and other commonly used tags.

Expression Analysis

A master plate of positive clones will be produced. The clones will be used to transform an expression strain. Overnight cultures of 1 ml will be used to produce glycerol stocks as well as inoculate 0.5 ml cultures for expression trials. After the expression incubation the cells are lysed by chemical additive and target protein is partially purified by metal affinity chromatography in 96-well plate format on the Biomek. Elutions are analyzed for the protein of interest using SDS-PAGE analysis. The glycerol stocks of the clones in the expression strain will be handed off along with all the protocols necessary for reproducing these results at larger scale. The master plate of positive clones will be made available if requested.

Consulting

We provide consulting on trouble-shooting expression problems, review data and protocols, do limited bioinformatic analysis, provide HTP protocols, suggest possible experiments, help develop strategy and, in some cases, run limited trials and analysis.

Baculovirus Services

Expression cultures are set up in deep-well, round-bottomed, 24-well plates. The MOI is set using the titering information generated by QPCR. Incubation times and other parameters are varied for optimization. Target protein is partially purified by metal affinity chromatography in 96-well plate format on the Biomek. Elutions are analyzed for the protein of interest using the Labchip 90. The data relating to expression and purification yields will be given to the requesting lab. The virus, bacmids and all the protocols necessary for reproducing these results at larger scale will be transferred to the requesting lab. The master plate of positive clones will be made available if requested.

Mammalian services

Transient transfection will be used for expression testing. The cultures will be seeded in 5 ml volumes in 24-well blocks. This will be done in duplicate or triplicate to compare expression at 24-, 48- and 72-hour incubations. If necessary, longer time-points may be examined. The target protein is partially purified by metal affinity chromatography in 96-well plate format on the Biomek. Elutions are analyzed for the protein of interest using SDS-PAGE analysis. The data relating to expression and purification yields will be given to the requesting lab. The plasmid DNAs and all protocols will be transferred to the requesting lab.

Resources

The HTP lab has created several series of vectors for use in bacterial expression.

These vectors are primarily designed to be compatible with Ligation-Independent Cloning (LIC), which makes them useful for high-throughput parallel cloning.

Each series of vectors makes use of replication origins from different incompatibility groups in E. coli, which allows them to be used in combination with one another. Each series has a set of fusion tags and promoter combinations to provide for maximum flexibility in expression trial design. Additionally, the CDF and RSF series of vectors were constructed to be relatively small so they can be used to clone very large genes or multiple-gene cassettes.