title: Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction.
abstract: Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 A resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.
authors.affiliation: Department of Molecular Biology, University of Geneva, Geneva, Switzerland.
authors.forename: Soheila
authors.initials: S
authors.lastname: Emamzadah
authors.forename: Tom J
authors.initials: TJ
authors.lastname: Petty
authors.forename: Victor
authors.initials: V
authors.lastname: De Almeida
authors.forename: Taisuke
authors.initials: T
authors.lastname: Nishimura
authors.forename: Jacques
authors.initials: J
authors.lastname: Joly
authors.forename: Jean Luc
authors.initials: JL
authors.lastname: Ferrer
authors.forename: Thanos D
authors.initials: TD
authors.lastname: Halazonetis
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D003460:Crystallization; D003516:Cycloparaffins / Q000737:chemistry* / Q000378:metabolism; D023281:Genomics; D006801:Humans; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D029681:Arabidopsis Proteins; D003516:Cycloparaffins; D009687:Nuclear Proteins; D011108:Polymers; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D009113:Muramidase; D014357:Trypsin; D000074183:ATPases Associated with Diverse Cellular Activities; C409208:MOM protein, Arabidopsis
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