RESUMO
SEPALLATA3 (SEP3) can be attributed to E class gene of the ABCE model of floral organ development. In order to reveal how SEP3 proteins form polymers, and the relationship between the polymers and their biological functions, the experiments of Arabidopsis thaliana AtSEP3 protein soluble expression in vitro were performed to construct a vector of prokaryotic expression, and investigate induced expression of recombinant proteins in Escherichia coli cells. The protein soluble expression was analyzed through the aspects of different protein domains, induction time, induction temperature, etc. Different structural domains and expression conditions were screened, and 0.1% IPTG inducing at 22 oC for 15 h was estimated as an optimal expression strategy. The nickel chelating resin was used to purify the protein in size exclusion chromatography (SEC) and the results indicated that AtSEP3 protein was present in the form of tetramer.
RESUMO
CpfS1 Gene cloned from arabidopsis thaliana was expressed in Escherichia coli DH5α. A cDNA fragment about 320 bp was amplified from the total RNA of arabidopsis thaliana seeds by reverse transcription PCR (RT-PCR) with a pair of specific primers based on the sequences of the AtCpfS1 gene. The recombinant prokaryotic expression vector pET30a-AtCpfS1 was constructed by inserting the cDNA fragment encoding the mature peptide into the prokaryotic expression vector pET30a, and then transformed into E. coli DH5α. Sequence analysis showed that the fragment length was 346 bp containing a full coding region of 332 bp encoding 76 amino acid residues with a molecular mass of 21.5 kD. The SDS-PAGE electrophoresis analysis showed that the best expression was induced by 21oC and 3.6×10-3 mol/L IPTG, under which a relative molecular weight of 82.5 kD recombinant protein was produced. The nickel chelating resin was used to purify the protein in size exclusion chromatography (SEC) and the results indicated that AtCpfS1 protein was present in the form of tetramer.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Homeodomínio/genética , Sementes/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Plasmídeos/química , Plasmídeos/metabolismo , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/metabolismo , Fatores de Transcrição/metabolismoRESUMO
We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (microFACS) for sorting various biological entities. Compared with conventional FACS machines, the microFACS provides higher sensitivity, no cross-contamination, and lower cost. We have used microFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors. Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E. coli cells and shown that the bacteria are viable after extraction from the sorting device. These sorters can function as stand-alone devices or as components of an integrated microanalytical chip.
