Extracellular Expression, Purification, and Characterization of a Winter Flounder Antifreeze Polypeptide from Escherichia coli

Li, Tong; Lin, Qingsong; Wong, Wan Keung; Ali, Asma; Lim, Daniel; Sung, Wing L.; Hew, Choy L.; Yang, Dan

doi:10.1006/prep.1999.1176

Cited by 34 publications

(20 citation statements)

References 24 publications

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“…41 All of these constructs, however, are not structurally identical to the native isoform, and as such they are cannot achieve the same level of TH activity or change ice crystal morphology in the same way. Here, we created a recombinant expression system for fully native, isotopically labeled, HPLC6 isoform to determine what effect the C-terminal amide and Arg 37 side chain have on the protein's structure and function.…”

Section: Discussionmentioning

confidence: 99%

“…Both rHPLC6-Ala 37 and rHPLC6-Arg 37 had very similar TH activities ($65% activity compared with the wild-type protein), which is close to the 70% activity reported by Tong et al for their recombinant HPLC6 isoform that also lacked a C-terminal amide. 41 At 3 mM protein concentration, there is an apparent difference in TH between rHPLC6-Arg 37 and rHPLC6-Ala 37 (14% less activity for the Ala mutant). Higher concentrations of the Ala 37 form undergo nonspecific aggregation as seen by increased linewidths in the 15 N-HSQC spectra (data not shown), such that the loss of activity arises from aggregated protein not being able to inhibit ice growth.…”

Section: Activity Of Type I Afp and C-terminal Mutantsmentioning

confidence: 96%

“…41 A description of the material and methods used for the cloning and preparation of the nonamidated protein is included in the Supporting Information.…”

Section: Cloning Expression and Protein Purificationmentioning

confidence: 99%

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Increased flexibility decreases antifreeze protein activity

Patel

Graether

2010

Protein Science

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Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single a-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg 37 HPLC6, nonamidated HPLC6 Ala 37 , amidated HPLC6 Ala 37 , and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still a-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala 37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface.

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Section: Discussionmentioning

confidence: 99%

Section: Activity Of Type I Afp and C-terminal Mutantsmentioning

confidence: 96%

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Increased flexibility decreases antifreeze protein activity

Patel

Graether

2010

Protein Science

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“…Our results demonstrate that AspSP construct without the signal peptide was present in the periplasm as well as in the extracelular medium, and with the signal peptide at negligible levels in the cytoplasm (Figure 2). Secreted proteins can leak from the periplasmic space into the culture medium possibly due to an increased permeability of the cell membrane (Tong et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Comparison between Two Erwinia carotovora L-Asparaginase II Constructions: cloning, Heterologous Expression, Purification, and Kinetic Characterization

Wink¹,

Bogdawa²,

Renard³

et al. 2010

J Microbial Biochem Technol

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L-Asparaginase II from Erwinia carotovora may represent an important alternative therapy in the treatment of acute childhood lymphoblastic leukaemia, despite its promising lower glutaminase activity than Escherichia coli and Erwinia chrysanthemi L-asparaginases II, currently used in treatment of this disease. Here we describe cloning, expression, purification and determination of steady-state kinetic parameters for E. carotovora L-asparaginase II: with (AspSP) and without the signal peptide (AspMP). AspMP was purified to homogeneity by a single-step protocol with 91% yield, and AspSP by a two-step protocol with 28% yield. In addition, both enzymes presented similar high specific activities: 208.1 and 237.6 U mg -1 , respectively. The K m and k cat values showed that AspMP has lower glutaminase activity than AspSP. Moreover AspMP is produced by a simpler purification protocol, and at higher yield. This process can be amenable to large scale production and be of interest to researchers and biopharmaceutical companies.

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“…In E. coli , proteins normally do not secreted into the extracellular circumstance except for a few classes of proteins such as toxin and hemolysin. However, small proteins are frequently released into the culture medium depends on the characteristics of signal sequences and proteins (Choi and Lee 2004; Tong et al 2000). Many studies have therefore been carried out to improve the secretion efficiency of recombinant proteins in E. coli expression systems (Baneyx and Mujacic 2004; Cornelis 2000; Klatt and Konthur 2012; Mergulhao et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli

Han¹,

Machhi²,

Berge³

et al. 2017

AMB Expr

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Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against Staphylococcus aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. Indeed, one of the novel signal peptides improved the yield of secreted ATH35L by 3.5-fold in a fed-batch fermentation process and at the same time maintained processing at the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0394-1) contains supplementary material, which is available to authorized users.

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Extracellular Expression, Purification, and Characterization of a Winter Flounder Antifreeze Polypeptide from Escherichia coli

Cited by 34 publications

References 24 publications

Increased flexibility decreases antifreeze protein activity

Increased flexibility decreases antifreeze protein activity

Comparison between Two Erwinia carotovora L-Asparaginase II Constructions: cloning, Heterologous Expression, Purification, and Kinetic Characterization

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli

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