Expression of an active recombinant lysine 49 phospholipase A2 myotoxin as a fusion protein in bacteria

Giuliani, Cesidio; Iemma, Mônica Rosas Costa; Bondioli, Ana Cristina Vigliar; Souza, Dulce Helena Ferreira de; Ferreira, Leonardo L. G.; Amaral, André Capaldo; Salvini, Tania F.; Selistre-de-Araújo, Heloísa Sobreiro

doi:10.1016/s0041-0101(01)00142-8

Cited by 18 publications

(11 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Too low expression yields are found when using eukaryotic host cells (Lefkowitz et al, 1999;Pernas et al, 1991;Tanaka et al, 1988;Valentin et al, 1999;van den Bergh et al, 1987). Attempts to express PLA 2 s in soluble form in E. coli by using signal sequences were successful with PLA 2 from Agkistrodon contortrix laticinctus (Hodgson et al, 1993) and Notechis scutatus scutatus (Giuliani et al, 2001) but failed with PLA 2 from Agkistrodon piscivorus piscivorus (Lathrop et al, 1992) and with bv-PLA 2 as shown in this study. With PLA 2 from porcine pancreas the enzyme yields obtained in this way were very low (De Geus et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

Production of synthetically created phospholipase A₂ variants with industrial impact

Markert

Mansfeld

Schierhorn

et al. 2007

Biotech & Bioengineering

View full text Add to dashboard Cite

Phospholipases A(2) (PLA(2)) play an important role for the production of lysophospholipids. Presently they are mainly obtained from porcine or bovine pancreas but these mammalian sources are not accepted in several fields of application. To make accessible a non-mammalian PLA(2) to industrial application, synthetic genes encoding PLA(2) from honey bee (Apis mellifera) with modified N-termini were constructed and expressed in Escherichia coli. While expression of the gene with an N-terminal leader sequence to direct the protein into the periplasm failed, four variants with slightly modified N-termini (I1A-PLA(2), I1V-PLA(2), His(6)-tagged PLA(2) and PLA(2) still containing the start methionine) were successfully expressed. In all cases, the PLA(2) variants were produced as inclusion bodies. Their protein content amounted to 26-35% of total cell protein. The optimized renaturation procedure and subsequent purification by cation-exchange chromatography yielded pure active enzymes in yields of 4-11 mg L(-1). The recombinant PLA(2) variants showed activities, far-UV CD and fluorescence spectra similar to the glycosylated PLA(2) isolated from the venom glands of honey bee (bv-PLA(2)). The thermodynamic stabilities of the recombinant enzymes calculated from the transition curves of guanidine hydrochloride induced unfolding were also nearly identical to the stability of bv-PLA(2). For the variant I1A-PLA(2) high-cell density fermentation in 10 L-scale using mineral salt medium was shown to increase the volumetric enzyme yield considerably.

show abstract

Section: Discussionmentioning

confidence: 99%

Production of synthetically created phospholipase A₂ variants with industrial impact

Markert

Mansfeld

Schierhorn

et al. 2007

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…The recovery of the mature rDE1, rDE2 and rG3 PLA 2 was 95, 133 and 10 mg/L culture, respectively. Giuliani et al 24 expressed lysine 49 PLA 2 as a fusion construct with maltose binding protein and tried to cleave the construct using a factor Xa protease. As the cleavage was unsuccessful, they finally obtained active PLA 2 as a fusion protein at a yield of 300 mg/L culture.…”

Section: Discussionmentioning

confidence: 99%

Recombinant expression and characterization of three group IB phospholipase A2 isoforms in the red sea bream Chrysophrys major

et al. 2003

View full text Add to dashboard Cite

We have previously cloned three distinct isoforms of group IB phospholipase A2 (PLA2), hepatopancreas DE‐1 and DE‐2 PLA2 and gill G‐3 PLA2, from the red sea bream Chrysophrys major and found that they are expressed in a tissue‐specific manner in the red sea bream. To understand the function of three group IB PLA2 isoforms, we constructed a bacterial expression system for DE‐1, DE‐2 and G‐3 PLA2. The cDNAs encoding for mature PLA2 were amplified by polymerase chain reaction (PCR) and subcloned in‐frame with a glutathione S‐transferase (GST) encoded by the vector pGEX‐4T‐1. The resulting plasmids were used to transform Escherichia coli BL21 (DE3) cells. Three recombinant PLA2 were expressed as a fusion protein with GST in E. coli cells by induction of Isopropyl‐1‐thio‐β‐D‐galactopyranoside. The bacterial cells were lyzed with strong alkaline solution and the fusion proteins were recovered as a soluble form. The fusion proteins were purified with affinity chromatography and cleaved by trypsin. Then, the recombinant DE‐1 and G‐3PLA2 were purified to near homogeneity by reversed‐phase high‐performance liquid chromatography (HPLC), and the recombinant DE‐2 PLA2 by ion exchange chromatography and reversed‐phase HPLC. Enzyme properties of purified recombinant DE‐1, DE‐2 and G‐3 PLA2 were found to be essentially the same as those of native PLA2.

show abstract

“…Attempts have been made to express the eukaryotic PLA 2 gene in Aspergillus by mutating the cysteines to increase the secretion (10). Researchers have also tried to express the eukaryotic PLA 2 in Escherichia coli with a fusion protein, but this approach requires the processing of protease to obtain a mature PLA 2 (11). Although yeast, fungal and bacteria expression systems can be used to produce the eukaryotic PLA 2 by fermentation, there are some difficulties in these systems, such as the structure of eukaryotic PLA 2 , which has many disulfide bonds that may influence the correct folding of the protein in E. coli, resulting in the formation of an inclusion body (12).…”

mentioning

confidence: 99%

“…Although yeast, fungal and bacteria expression systems can be used to produce the eukaryotic PLA 2 by fermentation, there are some difficulties in these systems, such as the structure of eukaryotic PLA 2 , which has many disulfide bonds that may influence the correct folding of the protein in E. coli, resulting in the formation of an inclusion body (12). Some studies have succeeded in generating soluble expression of the eukaryotic PLA 2 as a fusion protein, but attempts to cleave the fusion protein were unsuccessful (11). Additionally, the recombinant PLA 2 may be toxic to the E. coli and yeast because of its lipolytic activity (13,14).…”

mentioning

confidence: 99%

Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis

Wang

Zhang

Shi

2015

Journal of Bioscience and Bioengineering

View full text Add to dashboard Cite

Expression of an active recombinant lysine 49 phospholipase A2 myotoxin as a fusion protein in bacteria

Cited by 18 publications

References 24 publications

Production of synthetically created phospholipase A₂ variants with industrial impact

Production of synthetically created phospholipase A₂ variants with industrial impact

Recombinant expression and characterization of three group IB phospholipase A2 isoforms in the red sea bream Chrysophrys major

Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis

Contact Info

Product

Resources

About

Expression of an active recombinant lysine 49 phospholipase A2 myotoxin as a fusion protein in bacteria

Cited by 18 publications

References 24 publications

Production of synthetically created phospholipase A2 variants with industrial impact

Production of synthetically created phospholipase A2 variants with industrial impact

Recombinant expression and characterization of three group IB phospholipase A2 isoforms in the red sea bream Chrysophrys major

Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis

Contact Info

Product

Resources

About

Production of synthetically created phospholipase A₂ variants with industrial impact

Production of synthetically created phospholipase A₂ variants with industrial impact