Biomedical subjects

E Nordberg Karlsson

Publications and source records attributed to E Nordberg Karlsson.

6 recordsLinked to original sources

PubMed2005

A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor.

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.

Bioreactors↗

PubMed2004

A carbohydrate binding module as a diversity-carrying scaffold.

The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

Amino Acid Sequence↗

PubMed2002

Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3).

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.

Bacteriological Techniques↗

PubMed2001

Deletion of a cytotoxic, N-terminal putatitive signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus.

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 degrees C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 degrees C and unfolding temperatures up to 103 degrees C.

Base Sequence↗

PubMed2000

Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies.

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

Base Sequence↗

PubMed1997

Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus.

The gene (xyn1) encoding a Rhodothermus marinus xylanase has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xyn1 are repeated in tandem at the N-terminus and show similarity with the CBD family IV. The xyn1-gene is the first example encoding a CBD family IV in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10.

Amino Acid Sequence↗