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Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature.

Parrilli E, De Vizio D, Cirulli C, Tutino ML - Microb. Cell Fact. (2008)

Bottom Line: Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-beta-lactamase chimera.Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach.To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Chimica Organica e Biochimica, Università degli studi di Napoli Federico II - Complesso Universitario M,S, Angelo via Cinthia 4, 80126, Napoli Italia. tutino@unina.it.

ABSTRACT

Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic alpha-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity.

Results: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-beta-lactamase chimera.

Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic alpha-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.

No MeSH data available.


Related in: MedlinePlus

Psychrophilic α-amylase secretion in P. haloplanktis TAC125 gspE mutant strain. Western blotting analysis of extra-cellular media (lane 4) and corresponding cellular extract (lane 3) of P. haloplanktis TAC125 gspE mutant transformed with pFCamyΔCt plasmid. The western blotting analysis of extra-cellular media (lane 2) and corresponding cellular extract (lane 1) of recombinant P. haloplanktis TAC125-(pFCamyΔCt) recombinant cells is shown as a control.
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Figure 4: Psychrophilic α-amylase secretion in P. haloplanktis TAC125 gspE mutant strain. Western blotting analysis of extra-cellular media (lane 4) and corresponding cellular extract (lane 3) of P. haloplanktis TAC125 gspE mutant transformed with pFCamyΔCt plasmid. The western blotting analysis of extra-cellular media (lane 2) and corresponding cellular extract (lane 1) of recombinant P. haloplanktis TAC125-(pFCamyΔCt) recombinant cells is shown as a control.

Mentions: Secretion of psychrophilic α-amylase in P. haloplanktis TAC125 gspE mutant cells was studied. The mutant strain was transformed with pFCamyΔCt, a psychrophilic vector previously constructed for the recombinant secretion of α-amylase in the Antarctic bacterium [6]. The recombinant mutant strain was grown at 4°C till medium exponential phase and the α-amylase secretion was evaluated by Western blotting analysis of cellular (Figure 4, lanes 3) and extra-cellular (Figure 4, lanes 4) protein samples. The results demonstrated that the gspE mutation does not affect secretion of the psychrophilic enzyme.


Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature.

Parrilli E, De Vizio D, Cirulli C, Tutino ML - Microb. Cell Fact. (2008)

Psychrophilic α-amylase secretion in P. haloplanktis TAC125 gspE mutant strain. Western blotting analysis of extra-cellular media (lane 4) and corresponding cellular extract (lane 3) of P. haloplanktis TAC125 gspE mutant transformed with pFCamyΔCt plasmid. The western blotting analysis of extra-cellular media (lane 2) and corresponding cellular extract (lane 1) of recombinant P. haloplanktis TAC125-(pFCamyΔCt) recombinant cells is shown as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275215&req=5

Figure 4: Psychrophilic α-amylase secretion in P. haloplanktis TAC125 gspE mutant strain. Western blotting analysis of extra-cellular media (lane 4) and corresponding cellular extract (lane 3) of P. haloplanktis TAC125 gspE mutant transformed with pFCamyΔCt plasmid. The western blotting analysis of extra-cellular media (lane 2) and corresponding cellular extract (lane 1) of recombinant P. haloplanktis TAC125-(pFCamyΔCt) recombinant cells is shown as a control.
Mentions: Secretion of psychrophilic α-amylase in P. haloplanktis TAC125 gspE mutant cells was studied. The mutant strain was transformed with pFCamyΔCt, a psychrophilic vector previously constructed for the recombinant secretion of α-amylase in the Antarctic bacterium [6]. The recombinant mutant strain was grown at 4°C till medium exponential phase and the α-amylase secretion was evaluated by Western blotting analysis of cellular (Figure 4, lanes 3) and extra-cellular (Figure 4, lanes 4) protein samples. The results demonstrated that the gspE mutation does not affect secretion of the psychrophilic enzyme.

Bottom Line: Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-beta-lactamase chimera.Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach.To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Chimica Organica e Biochimica, Università degli studi di Napoli Federico II - Complesso Universitario M,S, Angelo via Cinthia 4, 80126, Napoli Italia. tutino@unina.it.

ABSTRACT

Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic alpha-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity.

Results: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-beta-lactamase chimera.

Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic alpha-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.

No MeSH data available.


Related in: MedlinePlus