Since 1993, the Canadian Space Agency (CSA) has been supporting Canadian scientists’ research on protein crystal growth in microgravity aboard six space missions. The most important of these missions was the CAPE project, which flew aboard space station Mir in 1997. Now, researchers are working on the PROSPECT project, which will be aboard the International Space Station in 2004. CSA has put together a user community made up of more than 15 research groups all across Canada. These research groups are studying the crystalline characteristics of proteins used in different applications in the field of biotechnology. In support of these missions, Canada obtained access to existing facilities aboard the US space shuttle, the Russian space station Mir and the International Space Station. In previous missions Canada made its presence felt and has become a leader in the field of protein crystal growth in microgravity.
Protein crystal growth is essential to the understanding of protein structure and function. Protein crystals grown in microgravity conditions can be larger and of better quality than crystals grown on Earth. These characteristics have a decisive effect on the study of their structure.
Indeed, this structural information, by allowing the rational development of therapeutic substances, enables researchers to produce better medication with fewer side effects. The crystalline characteristics of the proteins used are relevant to applications from cancer and diabetes treatments to the control of antibiotic-resistant bacteria.
The purpose of the Protein Crystal Growth (PCG) Program is to measure the effect of gravity on the crystal growth process. There are a number of techniques for growing protein crystals. However, a few specific techniques are used in space, such as liquid-liquid diffusion, vapour diffusion or dialysis. Liquid-liquid diffusion is defined as the direct contact of two fluids, that is, a protein solution and a precipitant solution, which, after equilibration, can form protein crystals. In the vapour diffusion technique, the fluids are not in direct contact, but crystallization occurs as the fluids evaporate. As in liquid-liquid diffusion, all necessary conditions must be met for crystallization to take place.
Before diffusion between fluids is permitted, a host of chemical, physical and biochemical characteristics must be controlled. For example, temperature, pH, pressurization, the concentration of the solutions and gravity are all elements that are relevant to the process of protein crystal growth. For the experiment to work in space, all the factors influencing crystal growth must be set on Earth, so that the only one left to measure is gravity.
The techniques mentioned above have all been used in space and have had varying degrees of success. However, short deadlines have meant that systematic comparative evaluations could not be done on the various crystal growth techniques used in space missions.
This is not unique to Canada, as each of the space agencies has had a similar experience. All we have to go on are the reports of experiments carried out aboard the spacecraft and unscientific data as to what worked and what didn’t. Mission data often relates to samples that are not statistically significant. However, according to the data gathered, about 30% of all proteins sent into space have formed crystals with good diffraction qualities, using a wide variety of apparatus. In about 15% of cases the space-grown proteins are of generally better quality than Earth-grown ones. In some cases the type of apparatus used limits our ability to do control experiments on Earth.
There are a number of ways of doing experiments on protein crystal growth. For example, if the technique used is liquid-liquid diffusion, two separate fluids must at a given moment be able to combine at an interface. Several methods are available to accomplish this in space. One way is to have sliding blocks bring the liquids into contact at the appropriate time. Another method involves placing each fluid in sequence in a tube and then freezing it; the result is two distinct layers of precipitant and protein. Then, in space, the fluids thaw and mix and crystal growth proceeds. However, this last method is less used by researchers since it limits their control over the experiment.
Most Earthbound crystal growth experiments are carried out by suspended drop crystallization in large matrices. This method is difficult to reproduce in a space-borne facility and, given the restrictions on mass and volume, most space experiments therefore make use of smaller matrices than those normally used on Earth. The materiel approaches used to date on space missions have been reviewed and none of them gives any clear direction as to the nature of the new Canadian facility to be sent up to the International Space Station. However, the approach taken must have clear objectives and requirements; financing should be provided for a design study that will solicit ideas for better approaches or for the presentation of innovative techniques.
Experiments carried out in the field of protein crystal growth: