Engineering Subtilisin into a Fluoride-Triggered Processing Protease Useful for One-Step Protein Purification†

Biochemistry ◽  
2004 ◽  
Vol 43 (46) ◽  
pp. 14539-14546 ◽  
Author(s):  
Biao Ruan ◽  
Kathryn E. Fisher ◽  
Patrick A. Alexander ◽  
Viktoriya Doroshko ◽  
Philip N. Bryan
Keyword(s):  
Protein Purification ◽  
One Step ◽  
Processing Protease

scholarly journals Protein purification from Arachis hypogaea in one step: stability studies and anticarcinogenic analysis

2019 ◽  
Vol 29 (1) ◽  
pp. 35-43
Author(s):  
Afaque Ahmad ◽  
Hirday N. Verma ◽  
Prahalad Bharti ◽  
Kamlesh Pandey ◽  
Shahbaz Khan ◽  
...  
Keyword(s):  
Protein Purification ◽  
Arachis Hypogaea ◽  
Stability Studies ◽  
One Step

scholarly journals Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

2020 ◽  
Vol 21 (2) ◽  
pp. 416
Author(s):  
Angel De La Cruz Pech-Canul ◽  
Javier Carrillo-Campos ◽  
María de Lourdes Ballinas-Casarrubias ◽  
Rosa Lidia Solis-Oviedo ◽  
Selena Karina Hernández-Rascón ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Purification ◽  
Phanerochaete Chrysosporium ◽  
Manganese Peroxidase ◽  
Expression System ◽  
White Rot Fungi ◽  
Functional Expression ◽  
White Rot ◽  
E Coli ◽  
One Step

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

scholarly journals Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope

2004 ◽  
Vol 55 (1) ◽  
pp. 135-147 ◽  
Author(s):  
Claus-Peter Witte ◽  
Laurent No�l ◽  
Janine Gielbert ◽  
Jane Parker ◽  
Tina Romeis
Keyword(s):  
Amino Acid ◽  
Protein Purification ◽  
Plant Material ◽  
One Step

One-Step Protein Purification Using Micropreparative Electrophoresis Fully Compatible with Protein Microsequencing

1994 ◽  
Vol 221 (2) ◽  
pp. 418-420 ◽  
Author(s):  
S. Desnoyers ◽  
S. Bourassa ◽  
M.H.P. West ◽  
G.G. Poirier
Keyword(s):  
Protein Purification ◽  
One Step
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