Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A single-stage fed-batch bioprocess for the production of a recombinant protein beta-galactosidase, by E. coli has been developed. The XL1-blue strain of E. coli which harbors a multi-number foreign plasmid PT was cultured in a reformulated medium. Critical medium components were selected and their respective concentrations were optimized with the Orthogonal Table method. An exponential substrate feeding schedule was used to maintain optimum conditions. Inhibition of growth and protein expression caused by excessive concentrations of glucose and acetate was investigated and subsequently minimized with an incremental nutrient feeding schedule which limited the specific growth rate of a culture. The program necessary to facilitate the control of substrate addition is fully described. This program has been used with a 2.5 l bioreactor and a commercially available software package for optimization without on-line or off-line measurement of optical density (OD), CO2, glucose or acetate. The optimized fed-batch process limited the acetate concentration to less than 20 mM; maintained an exponential growth phase for 50 h; and produced a cell density of 51 g l-1 dry cell weight (DCW) or 154 OD600 with a beta-galactosidase activity of 990 U ml-1.
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PMID:Optimization of a cultivation process for recombinant protein production by Escherichia coli. 136 46

The late genes of the temperate phage of Pseudomonas aeruginosa are organized in an analogous fashion to the corresponding transcription units of the Escherichia coli P2 and P2-like phages. Sequence analysis of four putative late promoter regions, PP(phiCTX), PO(phiCTX), PV(phiCTX) and PF(phiCTX), reveals no similarity to sigma(70)-type promoters or promoter consensus sequences found in Pseudomonas, indicating the apparent need for a phage-encoded protein to control the expression of phiCTX late genes. To elucidate the mode of expression of the late genes, we fused the putative late promoter regions to the promoterless lacZalpha gene, which encodes the N-terminal part of beta-galactosidase as a reporter enzyme, in the promoter-probe vector pME4510. The candidate transactivator gene orf34 was cloned into expression vector pHA10, to generate the plasmid pHA34. The two recombinant plasmids were introduced together into E. coli XL1-Blue and P. aeruginosa PAO1S-Lac. Our results demonstrate that in phiCTX three late promoters (PP(phiCTX), PO(phiCTX), and PF(phiCTX)) are activated upon induction by IPTG in PAO1S-Lac carrying the cloned promoters and pHA34. Deletions and base-pair substitutions obtained by PCR-mediated mutagenesis demonstrated that two conserved sequences, TTGTAG-N(9)-cTACAa and GcCGCGCGCGCGgC, are essential for effective late gene expression. Whereas the late promoters were active in P. aeruginosa, only weak beta-galactosidase activity was obtained in E. coli.
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PMID:Organization and activation of the late promoters of phiCTX, a cytotoxin-converting phage from Pseudomonas aeruginosa. 1191 13

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.
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PMID:Interplay of SOS induction, recombinant gene expression, and multimerization of plasmid vectors in Escherichia coli. 1220 89