The High-Throughput Protein Lab (HTP) lab in the Center for Structural Biology can increase your “bandwidth” to accelerate discovery.
The primary service provided by the HTP is to help you obtain the protein you need to enable your research. We accomplish this through 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.
We have put in place processes for high-throughput cloning and expression testing in bacterial and baculovirus-infected insect cell systems and are developing an analogous process for mammalian systems. 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.
Bacterial projects can be processed in multiples (or subsets) of 96, baculovirus projects in multiples of 24 (up to 48 at a time). The average length of expression testing is 3-5 weeks for bacteria, 6-8 weeks for baculovirus. The 24-well block format is also being adapted for use with mammalian cells. Clonemanager software is employed for design of oligos and additional sequences to obtain optimal efficiency in PCR production. PCR reactions, LIC reactions, transformations, plating and plasmid minipreps are all carried out in multi-well formats (24- or 96-well) using a Biomek FX liquid handler. Both DNA and protein sample analysis in 96-well format is accomplished with a Caliper Labchip 90 instrument.
At the heart of our process lie the vector libraries that we have constructed. Our bacterial process began with vectors developed at the MCSG but 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 and four different promoters (>60 in all) to afford the greatest degree of flexibility in experimental design. An initial set of mammalian cytosolic expression vectors based on pCDNA3.1 have also been constructed with the same LIC region and fusions. This allows us to clone a single gene or fragment into many bacterial, baculovirus and (soon) mammalian expression vectors and carry out expression testing simultaneously.
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 have recently obtained 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.
The HTP lab has deep expertise and decades of experience in cloning and making eukaryotic and prokaryotic proteins. Let us enable your next project, for internal U-M recharge rates click here. All others outside of U-M should contact wclayb [at] umich.edu for rates.