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Redbull83
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on 7/9/2007 19:11 PM   
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can anyone help me please through the steps of tetracyclines production and the scale up process to produce this antibiotic.
thanx in advance.


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Tony Rook
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Refer to this Scientist Solutions Drug Manufacturing Post for some references.

Tony Rook



Tony Rook
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Here are some additional resources that you may find helpful:

Guide to Biotechnology from Biotechnology Industry Organization.



Glycosylation in Scale-up of Antibody Production. GEN Vol. 22, No. 12, June 15, 2002.



The Antibody Resource Page



Czirbik, Rudolf J., Steven M. Rosen, et al. Factors affecting antibody production efficiency in hollow-fiber bioreactors. Published in IVD Technology Magazine in July, 1996. (accessed June 11, 1997).



Jez Wayte. Optimization and Scale-upof Protein-free Processesfor Antibody Production. Lonza Biologics, 2002.



Cell Culture Scale-Up. Mammalian Cell Line Development,
Scale-Up and Productivity. Waltham, Massachusetts, USA. September 24-26, 2007.

Designed for cell culture personnel in production as well as process and cell line development, this meeting will feature case histories from the industrys leading product developers. We will cover the key aspects of cell line development, banking, and characterization; plus critical scale-up and productivity issues such as inoculum preparation, media formulation, feed and harvest strategies, oxygen delivery, and process control optimization for a wide range of biological products including antibodies, recombinant proteins, and viruses.



Theodore J Torphy. Pharmaceutical biotechnologyMonoclonal antibodies: boundless potential, daunting challenges. Current Opinion in Biotechnology 2002, 13:589591

This volume of Current Opinion in Biotechnology provides an up-to-date overview of the technologies used to discover and optimize new therapeutic mAbs, the application of mAbs to three specific therapeutic areas (oncology, immune-mediated inflammatory diseases and infectious diseases), and the
implementation of new manufacturing technologies. Woven throughout these reviews is the common theme of a technology platform in transition from promising concept to tangible reality.



Eva Jordan, Michael Hust, Andreas Roth, Rebekka Biedendieck, Thomas Schirrmann, Dieter Jahn and Stefan Dübel. Production of recombinant antibody fragments in Bacillus megaterium. Microbial Cell Factories 2007, 6:2
doi:10.1186/1475-2859-6-2

Abstract
Background: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the
periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.
Results: The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv.
Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli.
Conclusion: High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.

Tony Rook



Redbull83
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Posted By Redbull83
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Thank you very much for your help.



Tony Rook
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Were any of these references of much help to you? Hopefully, you get the full-text of the most pertinent references and then start digging through their reference lists. This usually leads me in the right direction.

Good luck!

Tony Rook



Redbull83
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Posted By Redbull83
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Tony Rook said:


Were any of these references of much help to you? Hopefully, you get the full-text of the most pertinent references and then start digging through their reference lists. This usually leads me in the right direction.

Good luck!


I have found this article which i think will be very useful however i need to buy access to it. The article is:

Antibiotic production by Streptomyces aureofaciens using self-cycling fermentation
Michael G. Zenaitis, David G. Cooper
Biotechnology and Bioengineering 1959-1995
Volume 44, Issue 11 , Pages1331 - 1336
Copyright 1994 John Wiley & Sons, Inc.



Tony Rook
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Posted By Tony Rook
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Here are a few more references that you may find useful:


Dairi T, Nakano T, Aisaka K, Katsumata R, Hasegawa M. Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline. Biosci Biotechnol Biochem. 1995 Jun;59(6):1099-106. PMID: 7612997

Abstract:
Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated. The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study. The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chl gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively. A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland. By Northern blot analysis, these three genes were suggested to be polycistronically transcribed.




Dairi T, Aisaka K, Katsumata R, Hasegawa M. A self-defense gene homologous to tetracycline effluxing gene essential for antibiotic production in Streptomyces aureofaciens. Biosci Biotechnol Biochem. 1995 Oct;59(10):1835-41. PMID: 8534971

Abstract:
By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis. The analysis of the nucleotide sequence of the 2.8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.




Reynes JP, Calmels T, Drocourt D, Tiraby G. Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus.
J Gen Microbiol. 1988 Mar;134(3):585-98. PMID: 3053973

Abstract:
Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.




Ohnuki T, Katoh T, Imanaka T, Aiba S. Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes. J Bacteriol. 1985 Mar;161(3):1010-6.

Abstract:
Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline.




Nakano T, Miyake K, Endo H, Dairi T, Mizukami T, Katsumata R.
Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens. Biosci Biotechnol Biochem. 2004 Jun;68(6):1345-52. PMID: 15215601

Abstract:
For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.




Sezonov GV, Isaeva LM, Lomovskaia ND. [Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens]
Antibiot Khimioter. 1990 Dec;35(12):7-11. Russian.
PMID: 2127667

Abstract:
The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.




Sharma A, Padwal-Desai SR. Biogenesis of Some Antibiotics in the Presence of 2-Chloroethylphosphonic Acid. Appl Environ Microbiol. 1986 Oct;52(4):605-6.

Abstract:
2-Chloroethylphosphonic acid (CEPA) affected both the growth of and antibiotic production in Streptomyces aureofaciens, S. griseus, S. antibioticus, and Penicillium citrinum. Streptomyces strains seemed to be more sensitive to the presence of CEPA in the medium than did the fungus. A decrease in both growth and antibiotic production was observed with a concomitant increase in the concentration of the ethylene-releasing compound in the medium. Higher concentrations of CEPA completely inhibited the growth of the above microorganisms.




Li XM, Novotná J, Vohradsk J, Weiser J. Major proteins related to chlortetracycline biosynthesis in a Streptomyces aureofaciens production strain studied by quantitative proteomics. Appl Microbiol Biotechnol. 2001 Dec;57(5-6):717-24.

Abstract:
Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed in cultures grown in the presence of benzyl thiocyanate, a specific stimulator of CTC biosynthesis used in industrial fermentations. The expression kinetics of the selected proteins was assessed using the technique of computer-assisted image analysis with the EQIAS software and the elongation factor Tu as an internal standard. Interestingly, the kinetic profiles were generally not identical. including those of anhydrotetracycline monooxygenase and the 13-kDa subunit of tetracycline dehydrogenase, two enzymes involved, in the terminal sequential steps of the CTC biosynthetic pathway. The presence of more forms of these enzymes with different charge characteristics was observed. The data presented demonstrated how dramatically the industrial microorganism can change its protein repertoire during the production phase; at least five proteins were nearly comparable in level to the most prominent proteins, exemplified by elongation factor Tu.



Tony Rook



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