scholarly journals Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli

2009 ◽  
Vol 74 (6) ◽  
pp. 607-618 ◽  
Author(s):  
Mira Milisavljevic ◽  
Drazen Papic ◽  
Gordana Timotijevic ◽  
Vesna Maksimovic
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Inclusion Bodies ◽  
Bond Formation ◽  
Aspartic Protease ◽  
The Other ◽  
Fusion Partner ◽  
Disulfide Bond Formation ◽  
Insoluble Protein ◽  
Successful Production

Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality.

scholarly journals The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Brian M. Meehan ◽  
Cristina Landeta ◽  
Dana Boyd ◽  
Jonathan Beckwith
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Anaerobic Growth ◽  
Strictly Anaerobic ◽  
Disulfide Bond Formation ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Laboratory Conditions

ABSTRACT Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited. IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

scholarly journals Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus

2000 ◽  
Vol 350 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Zhen-Zhong ZHANG ◽  
Satoru NIRASAWA ◽  
Yoshiaki NAKAJIMA ◽  
Michiteru YOSHIDA ◽  
Kiyoshi HAYASHI
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Region ◽  
Protein Expression ◽  
Inclusion Bodies ◽  
Active Enzyme ◽  
Vibrio Proteolyticus ◽  
Show Evidence ◽  
Inhibited Enzyme

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

scholarly journals Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1

2006 ◽  
Vol 397 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Li-Chiun Lee ◽  
Ya-Lin Lee ◽  
Ruey-Jyh Leu ◽  
Jei-Fu Shaw
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Water Molecule ◽  
Acyl Chain ◽  
Residual Activity ◽  
Catalytic Triad ◽  
The Other ◽  
Oxyanion Hole

Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75–80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.

scholarly journals Identification and Functional Analysis of an Immunoreactive DsbA-Like Thio-Disulfide Oxidoreductase of Ehrlichia spp.

2002 ◽  
Vol 70 (5) ◽  
pp. 2700-2703 ◽  
Author(s):  
Jere W. McBride ◽  
Lucy M. Ndip ◽  
Vsevolod L. Popov ◽  
David H. Walker
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Ehrlichia Canis ◽  
Disulfide Bond Formation ◽  
Ehrlichia Chaffeensis ◽  
Disulfide Oxidoreductase

ABSTRACT Novel homologous DsbA-like disulfide bond formation (Dsb) proteins of Ehrlichia chaffeensis and Ehrlichia canis were identified which restored DsbA activity in complemented Escherichia coli dsbA mutants. Recombinant Ehrlichia Dsb (eDsb) proteins were recognized by sera from E. canis-infected dogs but not from E. chaffeensis-infected patients. The eDsb proteins were observed primarily in the periplasm of E. chaffeensis and E. canis.

scholarly journals Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

2020 ◽  
Vol 47 (8) ◽  
pp. 563-571
Author(s):  
Hongmei Han ◽  
Weizhu Zeng ◽  
Guoqiang Zhang ◽  
Jingwen Zhou
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Industrial Application ◽  
Enzyme Activities ◽  
Inclusion Bodies ◽  
Carboxyl Terminus ◽  
Tyrosine Phenol Lyase ◽  
Functional Peptides ◽  
Important Enzyme ◽  
Aggregate Particles

Abstract The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.

Sign in / Sign up

Export Citation Format

Share Document