scholarly journals A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

2012 ◽  
Vol 13 (3) ◽  
pp. 3549-3562 ◽  
Author(s):  
Hussin A. Rothan ◽  
Ser Huy Teh ◽  
Kamariah Haron ◽  
Zulqarnain Mohamed
Keyword(s):  
Escherichia Coli ◽  
Pichia Pastoris ◽  
Comparative Study ◽  
Functional Characterization

scholarly journals Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Travis J. Wiles ◽  
Elena S. Wall ◽  
Brandon H. Schlomann ◽  
Edouard A. Hay ◽  
Raghuveer Parthasarathy ◽  
...  
Keyword(s):  
Comparative Study ◽  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Functional Characterization ◽  
Symbiotic Bacteria ◽  
Bacterial Isolates ◽  
Major Barrier ◽  
Allelic Exchange ◽  
Genetic Techniques

ABSTRACTCorrelating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis andin vitroassays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCEA great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product

Biochemistry ◽  
1993 ◽  
Vol 32 (41) ◽  
pp. 11112-11117 ◽  
Author(s):  
Lam H. Nguyen ◽  
Debra B. Jensen ◽  
Nancy E. Thompson ◽  
Daniel R. Gentry ◽  
Richard R. Burgess
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Functional Characterization ◽  
Rpos Gene

scholarly journals Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0145419 ◽  
Author(s):  
Mengmeng Dai ◽  
Changming Yu ◽  
Ting Fang ◽  
Ling Fu ◽  
Jing Wang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Pichia Pastoris ◽  
Human Platelet ◽  
Functional Characterization ◽  
Platelet Derived Growth Factor
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