Synthetic Gene DataBase
 

Synthetic Gene 40


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID3440
GenBank AccessionX70354SCO295616
GenBank GI4920510638023
Gene NamelipB (helper protein)lipB (helper protein)
Gene Length (bp)10621062
SpeciesBurkholderia glumae (Chromobacterium viscosum)Escherichia coli
StrainsBL21 (DE3)
CDSatggcgcaggccgatcgtccggcgcgcggcgggctggccgcgcgcccgatgcgcggcgcg
tcgttcgcgctggccgggctcgtcgcgtgtgccgcctgtgccgcggtcgtgctgtggctt
cggcccgccgccccgtcgcccgcgccggccggcgccgtcgcgggcgggccggcggccggc
gtgcccgccgcggcaagcggcgcggcggaggccgccatgccgttgccggcggcgctgccg
ggcgcgctggctggctcgcatgcgccgcgcctgccgctggccgccggcggccggctcgcg
aggacgcgcgcggtgcgcgagttcttcgactattgcctgaccgcgcagggcgaactgacg
cccgccgcgctcgatgcgctggtgcggcgcgagatcgccgcgcagcttgacggcagcccc
gcgcaagcggaggcgctcggcgtctggcgccgctatcgcgcctacttcgacgcgctcgcg
caattgcccggcgacggcgcggtgctcggcgacaagctcgatccggccgccatgcagctc
gcgctcgatcagcgcgcggcgctggccgaccgcacgctcggcgagtgggccgagccgttc
ttcggcgacgagcagcgccggcagcgccatgacctcgaacggatccggatcgccaacgac
accacgctgagccctgagcagaaggccgcgcggcttgccgcgctcgacgcgcagctgacg
ccggacgagcgcgcgcagcaggcggcgctgcatgcgcagcaggacgcggtgacgaagatc
gccgacttgcagaaggccggcgcgacgcccgaccagatgcgcgcgcagatcgcgcagacg
ctcgggcccgaggcggccgcgcgcgccgcgcagatgcagcaggacgacgaggcgtggcag
acgcgctatcaagcctatgcggccgagcgcgaccggatcgcggcgcaggggctcgcgccg
caggatcgcgatgcgcggatcgcgcagctcaggcagcagactttcacggcgccgggggag
gcgatccgcgcggcgtcgctcgatcgcggcgcgggcggttag
atggcacaggctgaccgcccggcccgcggtggtctggcagcacgcccgatgcgtggtgca
tctttcgctctggctggcctggttgcttgcgctgcttgcgcagcagttgttctgtggctg
cgtccggctgctccgagtccggctccggcgggtgcagttgcaggtggcccggcagcaggt
gttccggcagcagcttccggcgcagcagaagctgcgatgccgctgccggctgctctgccg
ggtgcactggctggttctcatgctccgcgtctgccgctggcagctggcggccgcctggca
cgtacccgtgcggttcgtgagttcttcgactactgcctgactgcgcagggtgaactgacc
ccggcggctctagacgcactggttcgtcgtgaaatcgctgcacagctggacggctctccg
gcacaggcagaagctctgggtgtttggcgccgctaccgtgcttacttcgacgcactggct
cagctgccgggcgatggtgcagtactgggtgacaaactggacccggctgctatgcagctg
gctctggaccagcgtgcagctctggctgaccgtaccctgggtgaatgggcagaaccgttc
ttcggcgacgaacagcgccgtcagcgtcacgacctggaacgtatccgtatcgcgaacgac
accactctgtccccggaacagaaagctgctcgcctggcagcactggatgctcagctgacc
ccggacgaacgtgcgcagcaggcagcactgcatgctcagcaggacgctgtaactaaaatc
gctgaccttcagaaagcaggtgctaccccggaccagatgcgtgctcagatcgctcagacc
ctgggcccggaagcagcagctcgtgcagcgcagatgcagcaggacgacgaagcatggcag
acccgttaccaggcttacgcagcagaacgcgatcgcatcgctgcgcagggtctggctccg
caggaccgtgatgcacgtatcgctcagctgcgccagcagactttcaccgcaccgggtgaa
gcaatccgtgcagcttccctggaccgtggcgctggtggttaa
5' End
3' End
Notes
Expression VectorNApET20b(+)
Assay MethodsNASDS-PAGE
ResultsExpression not determinedThe full-length lipB gene was not observed in SDS-PAGE analysis but the truncated gene d79lipB revealed expression levels of 25%.
Protein FunctionHelper protein for lipA; Chaperone
Recoding PurposeTo improve expression
Synthesized ByAuthors
Recoding MethodNative Chromobacterium viscosum optimized for codon usage and expression in Escherichia coli
Publication Author(s)Traub PC, Schmidt-Dannert C, Schmitt J, Schmid RD.
Corresponding AuthorRolf. D. Schmid
Corresponding AddressInstitut fur Technische Biochemie, Universitat Stuttgart, Germany.
Publication Year2001
Publication TitleGene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp.KWI 56 and Chromobacterium viscosum lipases and their chaperones.
AbstractPseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.
JournalAppl Microbiol Biotechnol.. 55(2): 198-204.
SummaryLipase and its helper protein (chaperone) in Pseudomonas sp. and C. viscosum were codon optimized for expression in E. coli (rare codons to optimal codons). It appeared that none of the full-length gene could be over-expressed. Interestingly, removing the 5' sequence or replacing it with another leader sequence (e.g. ompA) led to significant increase in expression. This suggests that codon optimization cannot solve the posttranslational problems.
Comments
Discussion http://www.evolvingcode.net/forum/viewtopic.php?t=494
PubMed ID11330714
Submitter NameWu, Gang
Submitter AddressDepartment of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 USA
Entry ConfirmationNo
 
 

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