Refolding and activation of recombinant N-carbamoyl-d-amino acid amidohydrolase from Escherichia coli inclusion bodies

2005 ◽  
Vol 40 (6) ◽  
pp. 2135-2141 ◽  
Author(s):  
Hsiu-Mei Chen ◽  
Kun-Ying Lin ◽  
Chi-Hung Lu
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Inclusion Bodies ◽  
Acid Amidohydrolase

Production of D-P-HYDROXYPHENYLGLYCINE BY N-CARBAMOYL-D-amino Acid Amidohydrolase-Overproducing Escherichia coli Strains

1999 ◽  
Vol 15 (4) ◽  
pp. 603-607 ◽  
Author(s):  
Y.-P. Chao ◽  
T.-Y. Juang ◽  
J.-T. Chern ◽  
C.-K. Lee
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Acid Amidohydrolase

scholarly journals Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

2007 ◽  
Vol 402 (3) ◽  
pp. 429-437 ◽  
Author(s):  
Shimin Jiang ◽  
Chunhong Li ◽  
Weiwen Zhang ◽  
Yuanheng Cai ◽  
Yunliu Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Protein Solubility ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Triple Mutant ◽  
E Coli ◽  
Acid Amidohydrolase

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

Production of thermotolerant N-carbamyl-d-amino acid amidohydrolase by recombinant Escherichia coli

1999 ◽  
Vol 87 (2) ◽  
pp. 149-154 ◽  
Author(s):  
Hirokazu Nanba ◽  
Yasuhiro Ikenaka ◽  
Yukio Yamada ◽  
Kazuyoshi Yajima ◽  
Masayuki Takano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Escherichia Coli ◽  
Acid Amidohydrolase

A novel N-carbamoyl-l-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-l-cysteine amidohydrolase expressed in Escherichia coli

2004 ◽  
Vol 65 (6) ◽  
pp. 686-693 ◽  
Author(s):  
Tetsuo Ohmachi ◽  
Megumi Narita ◽  
Maki Kawata ◽  
Akiko Bizen ◽  
Yoshiharu Tamura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Pseudomonas Sp ◽  
Purification And Characterization ◽  
Acid Amidohydrolase

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

Fate of MS2 proteins synthesized in Escherichia coli exposed to amino acid analogues.

1975 ◽  
Vol 16 (4) ◽  
pp. 1090-1093 ◽  
Author(s):  
W F Prouty
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Analogues

Conditions Resulting in Formation of Properly Assembled Retroviral Capsids within Inclusion Bodies of Escherichia coli

1999 ◽  
Vol 64 (8) ◽  
pp. 1348-1356 ◽  
Author(s):  
Michaela Rumlová-Kliková ◽  
Iva Pichová ◽  
Eric Hunter ◽  
Tomáš Ruml
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Amorphous Material ◽  
Bacterial Expression ◽  
Biologically Active ◽  
Capsid Assembly ◽  
Cultivation Medium ◽  
Viral Particles ◽  
Induction Temperature

It has been generally accepted that inclusion bodies (IBs) formed in Escherichia coli consist of non-biologically active aggregated proteins, which are stabilized by non-productive interactions. We show here that bacterial expression of a retroviral capsid polyprotein results in formation of insoluble IBs that contain fully assembled viral particles connected with amorphous material. The efficiency of IBs formation and capsid assembly was not significantly affected by changes in induction temperature, pH of cultivation medium or the level of expression.

scholarly journals Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.

1994 ◽  
Vol 269 (51) ◽  
pp. 32708
Author(s):  
T G Huang ◽  
J Suhan ◽  
D D Hackney
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Position ◽  
Motor Domain ◽  
Kinesin Motor

scholarly journals Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli.

1988 ◽  
Vol 263 (28) ◽  
pp. 14276-14280 ◽  
Author(s):  
T Kawakami ◽  
Y Akizawa ◽  
T Ishikawa ◽  
T Shimamoto ◽  
M Tsuda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitutions
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