Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

Zocchi, Andrea; Jobé, Anna Marya; Neuhaus, Jean‐Marc; Ward, Thomas R.

doi:10.1016/j.pep.2003.09.001

Cited by 43 publications

(33 citation statements)

References 36 publications

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“…Heat-shock competent BL21(DE3)pLysS E. coli strain (Novagen-Juro, Luzern, Switzerland) were transformed by pET11b-SAV plasmid containing a gene encoding for recombinant streptavidin fused with the T7-tag [15][16][17][18]. Transformed bacteria were plated on Luria broth (LB) dishes [19] containing ampicilline (60 mg/mL), chloramphenicol (34 mg/mL) and glucose (1% w/v), and further incubated overnight at 377C.…”

Section: Production Of Recombinant Streptavidinmentioning

confidence: 99%

Electrophoretic behavior of streptavidin complexed to a biotinylated probe: A functional screening assay for biotin-binding proteins

2005

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Section: Production Of Recombinant Streptavidinmentioning

confidence: 99%

Electrophoretic behavior of streptavidin complexed to a biotinylated probe: A functional screening assay for biotin-binding proteins

2005

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“…Codon harmonization is important for the expression of foreign gene. For example, codon-usage modification resulted in higher avidin expression in E. coli [43] and P. pastries [44,45]. C. glutamicum belongs to the high G+C Gram-positive Actinobacteria, and C. glutamicum metK had 56.1% G+C content, whereas SAM2 had only 41.9% G+C content and may contain codons that are rarely used in C. glutamicum.…”

Section: Codon-optimized Sam2 Sam2-c Could Be Overexpressed and Itsmentioning

confidence: 99%

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

Han

Wang

2015

Biotech and App Biochem

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Two genes encoding methionine adenosyltransferase, SAM2 from Saccharomyces cerevisiae and metK from Corynebacterium glutamicum, were individually cloned into pDXW-8, the shuttle vector between Escherichia coli and C. glutamicum, and overexpressed in E. coli DH5α and C. glutamicum ATCC13032. In DH5α, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032, metK was overexpressed, its product MetK showed the enzyme activity and could convert l-methionine to S-adenosyl-l-methionine (SAM). However, when SAM2 was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM from l-methionine was observed. Reverse transcription PCR analysis and SDS-PAGE showed that SAM2 was transcribed but not translated in C. glutamicum. Therefore, SAM2-C, a mutant SAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convert l-methionine to SAM. Finally, SAM2-C and metK were individually overexpressed in E. coli BL21(DE3), and their products SAM2-C and MetK were purified and characterized. The optimum activity for both enzymes was found at pH 8.5 and 35 °C; SAM2-C and MetK have similar K for ATP, but quite different K for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.

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“…Pichia pastris, 24) and plants. 25,26) Here, we optimized the pattern of codon use by the tamavidin 2 gene for high-level expression in human cells.…”

Section: )mentioning

confidence: 99%

High-level expression of tamavidin 2 in human cells by codon-usage optimization

Takakura

Katayama

Nagata

2015

Bioscience, Biotechnology, and Biochemistry

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Tamavidin 2 is a fungal protein that binds to biotin with an extremely high affinity. Tamavidin 2 is superior to avidin or streptavidin in terms of its low-level non-specific binding and high-level thermal stability. However, the gene for tamavidin 2 is highly expressed in Escherichia coli but not in mammalian cells, restricting its application as an affinity tag in mammalian cells. Here, we optimized the codon usage of tamavidin 2 for human cells and found that the resultant mutant expressed tamavidin 2 at approximately 30-fold higher level compared with the native gene. The protein thus produced in human cells could be purified by iminobiotin affinity chromatography, bound tightly to biotin, and was stable at high temperature (82 °C). This powerful technology for high-level expression of tamavidin 2 in mammalian cells will be of value in evaluating various fusion proteins produced in mammalian cells for numerous applications.

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Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

Cited by 43 publications

References 36 publications

Electrophoretic behavior of streptavidin complexed to a biotinylated probe: A functional screening assay for biotin-binding proteins

Electrophoretic behavior of streptavidin complexed to a biotinylated probe: A functional screening assay for biotin-binding proteins

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

High-level expression of tamavidin 2 in human cells by codon-usage optimization

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