The challenges of protein expression, purification, and production have been identified and are being addressed in both heterologous and homologous systems.
The ultimate goal of the research effort dictates what approach can be brought to bear, whether that goal be proper crystallization for resolution of protein structure or production of therapeutic proteins for use in the clinic.
Understanding the full range of complexities for protein expression and production can only come by the exchange of ideas among colleagues and collaborators. This was the focus of the CHI’s “Bioprocessing Summit” last month.
Well-characterized protein expression systems include cell-free extracts, E. coli, S. cerevisiae, baculovirus-insect cell systems, CHO cells, and HEK293 cells. Interestingly, there is usually no universal choice of the best system to use to produce any particular protein. This typically needs to be determined empirically for each specific protein of interest.
The lab of Don Jarvis, Ph.D., professor of molecular biology at the University of Wyoming, is focused on optimizing the baculovirus-insect cell system for recombinant glycoprotein production. Protein glycosylation, which is important because it influences protein structure and function, involves three distinct steps: first, the transfer of the initial oligosaccharide precursor; second, trimming of some terminal sugars; and third, addition of different sugars to form the final oligosaccharide side-chain.
Glycosylation patterns are cell type dependent and insect and mammalian pathways are distinctly different. The Jarvis lab has been focused on identifying genes encoding the endogenous insect cell enzymes and then adding human genes to enable insect cells to synthesize human-type glycan structures.
“It turns out that insect cells are highly amenable to uptake and incorporation of DNA,” Dr. Jarvis indicated. “We have been able to co-transform Sf9 insect cells with up to seven unlinked markers plus a selectable marker. After selection, we can find clones that express all seven markers at surprisingly high frequency. Screening is particularly easy as the glycan-processing pathway is linear. Thus, we can verify the presence of terminal sialic acids in a simple and fast lectin binding assay, which confirms that all seven genes have been expressed.”
“We have found that a minimum of six genes are needed to humanize the insect cell glycosylation pathway. We are now focused on finding innovative ways to increase the efficiency of this biosynthetic process.”
Exploiting S. cerevisiae
At the University of Delaware Biotechnology Institute, Carissa Young, Ph.D., and her colleagues are asking the question, what’s preventing us from expressing our heterologous protein at high functional yields in yeast cells? The lab has taken a systematic approach to understand why proteins fail to make it to their final destinations, either the plasma membrane or secreted into the media.
To investigate protein-trafficking effects, the lab has designed expression cassettes that incorporate conventional affinity tags and fluorescent proteins used for the identification and purification of the heterologous proteins. This enables the development of rationally designed chimeras that can also be used to identify subcellular localization in numerous organelles including the ER, Golgi, and plasma membrane.
“We have taken a holistic approach to determine which elements of protein structure impact trafficking of heterologous proteins in yeast. Specifically, exit motifs play a pivotal role in ER-to-Golgi trafficking within the secretory pathway, and very few studies have assessed these effects,” shared Dr. Young.
“Using chimeric receptors, we confirmed at both the gene and protein levels the results of trafficking motifs and domain-swapping, while correlating these effects to growth rate, expression levels, and activity. Different from an ER-signal sequence located at the N-terminus, ER exit and retrieval motifs consist of a 3- to 4-amino acid sequence, generally clustered in groups of two or three at the C-terminus of the protein. Interestingly, most affinity tags used in protein work contain several trafficking motifs that can be exploited for heterologous protein expression.”
The lab is working with GPCR membrane proteins, members of the adenosine and neurokinin receptor families, as well as antibody fragments. Copy number, time of expression, and the induction and culture conditions can all impact the level of expression for the membrane protein or the secreted protein. Consequently, genes that encode these heterologous protein sequences are inserted directly into the yeast genome with a controlled copy number.
Time-course experiments evaluate samples, cells, and media in order to quantify the total level of protein expression and the degree to which antibody fragments are secreted. Expressed and purified protein levels are calculated by Western blots coupled with standards developed in-house.
Membrane Protein Expression
The challenge of overexpressing membrane proteins in cells needs to be addressed on multiple levels. First, can you make enough protein without killing the cells? Second, what detergent provides optimal extraction of the membrane protein and stabilizes the protein without aggregation? (Membrane proteins often display significant nonspecific aggregation.)
Third, concentration of the protein enables formation of the proper crystal lattice structure. At concentrations of 1 mg/mL the protein solution is so dilute that the probability of forming crystal nuclei (the initial event in crystallization) is extremely low. The majority of proteins crystallized have initial concentrations between 5 and 20 mg/mL.
At higher concentrations in homogeneous solutions the proteins have a higher probability of interacting in the proper orientation to form a proper crystal lattice (nucleation event). Unfortunately, for many aqueous and membrane proteins, as the concentration of purified protein is increased, there is often an increase in the formation of nonspecific aggregation producing a nonhomogeneous population of purified protein—not desirable for crystallization.
Self-interaction chromatography is a method that has been used to assess the best conditions to place a soluble protein that will support stability of the protein in high concentration without formation of aggregates. Development of an automated analytical self-interaction chromatography system is now being adopted for identification of buffer conditions that will support rapid identification of the optimum solubility conditions followed by identification of solution conditions with a higher probability of crystal formation.
“We’ve taken this same approach to determine the best buffer conditions to grow membrane protein crystals. We load the column with 0.75 mg of protein, allowing it to form covalent bonds to a polymer-based bead matrix in the column. This guarantees that every surface of the protein is exposed to the solvent,” said Larry DeLucas, Ph.D., director, Center for Structural Biology, University of Alabama at Birmingham.
“We then load a few µg of the same protein onto the column under 100 different buffer conditions that contain different solubilizing additives (excipients) and measure the elution retention time for the injected protein. This represents a small random sampling of the total number of possible combinations and concentrations that could be investigated.
“This experimental data along with other parameters, including total protein covalently bound to the matrix and the column void volume is input into an equation that calculates for each solution condition the second virial coefficient, a thermodynamic term that represents the sum of all protein-protein interactions. The calculated second virial coefficient values with their respective solution conditions are then input into an artificial neural network (ANN),” he continued.
“The ANN uses this input data to predict the second virial coefficient values for the complete factorial of possible solution conditions. The resulting predicted values provide an assessment of each solution condition’s ability to reduce (positive value) or increase (negative value) protein-protein interactions. For protein crystallization, we are looking for a gentle net attraction, solution parameters that force the protein molecules to display weak attraction, thereby improving the probability of forming high-quality crystals.”
From this approach the DeLucas lab has determined that lithium chloride in solutions up to 1 M can support the concentration of the cystic fibrosis transmembrane regulator protein from 0.05 mg/mL to 0.5 mg/mL in a homogeneous solution without formation of aggregates.
As a former payload specialist who flew aboard the 1992 U.S. Space Shuttle Columbia, mission STS-50, Dr. DeLucas was able to demonstrate that certain protein crystals are of higher quality when grown in a microgravity environment as compared with earth-grown crystals of the same protein. At that time, the challenge was the short duration of time in space.
Crystals grow slower in space and so the size of the crystals from the typical space shuttle 10-day mission was often too small for diffraction analysis. With the establishment of the International Space Station (ISS) the time constraints can be overcome. Dr. DeLucas will be contributing more than 100 proteins from researchers in academia and industry to a future commercial launch of the Space X Dragon laboratory, flown on the Falcon rocket.