Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy

N. Siren (Corresponding Author), J. Weegar, J. Dahlbacka, Nisse Kalkkinen, K. Fagervik, Matti Leisola, Niklas Von Weymarn

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

In the present paper we describe the cloning and extracellular expression of the HIV‐1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low‐temperature fed‐batch strategy for decreasing end‐product degradation by proteases. The nef gene in a pPICZαA vector was integrated into the genome of three different P. pastoris strains, namely X‐33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild‐type strain (X‐33) was found to be the best choice. The decreased end‐product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44‐fold utilizing the low‐temperature strategy compared with the standard fermentation. Purification of histidine‐tagged Nef was performed in one step using a Ni2+‐nitrilotriacetate–Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix‐assisted laser‐desorption ionization–time‐of‐flight MS, reversed‐phase HPLC and N‐terminal‐sequence analysis.
Original languageEnglish
Pages (from-to)151-158
Number of pages8
JournalBiotechnology and Applied Biochemistry
Volume44
Issue number3
DOIs
Publication statusPublished - 2006
MoE publication typeA1 Journal article-refereed

Fingerprint

Pichia
HIV-1
Peptide Hydrolases
nef Genes
Genes
Proteins
Degradation
Temperature
Fungal Proteins
Cloning
Yeast
Biomass
Fermentation
Purification
Methanol
Organism Cloning
Polyacrylamide Gel Electrophoresis
Western Blotting
High Pressure Liquid Chromatography
Genome

Keywords

  • Fed-batch
  • HIV-1 negative factor (HIV-1 Nef)
  • Methanol
  • Pichia pastoris
  • Protease
  • recombinant proteins

Cite this

Siren, N. ; Weegar, J. ; Dahlbacka, J. ; Kalkkinen, Nisse ; Fagervik, K. ; Leisola, Matti ; Von Weymarn, Niklas. / Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy. In: Biotechnology and Applied Biochemistry. 2006 ; Vol. 44, No. 3. pp. 151-158.
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abstract = "In the present paper we describe the cloning and extracellular expression of the HIV‐1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low‐temperature fed‐batch strategy for decreasing end‐product degradation by proteases. The nef gene in a pPICZαA vector was integrated into the genome of three different P. pastoris strains, namely X‐33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild‐type strain (X‐33) was found to be the best choice. The decreased end‐product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44‐fold utilizing the low‐temperature strategy compared with the standard fermentation. Purification of histidine‐tagged Nef was performed in one step using a Ni2+‐nitrilotriacetate–Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix‐assisted laser‐desorption ionization–time‐of‐flight MS, reversed‐phase HPLC and N‐terminal‐sequence analysis.",
keywords = "Fed-batch, HIV-1 negative factor (HIV-1 Nef), Methanol, Pichia pastoris, Protease, recombinant proteins",
author = "N. Siren and J. Weegar and J. Dahlbacka and Nisse Kalkkinen and K. Fagervik and Matti Leisola and {Von Weymarn}, Niklas",
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Siren, N, Weegar, J, Dahlbacka, J, Kalkkinen, N, Fagervik, K, Leisola, M & Von Weymarn, N 2006, 'Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy', Biotechnology and Applied Biochemistry, vol. 44, no. 3, pp. 151-158. https://doi.org/10.1042/BA20060001

Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy. / Siren, N. (Corresponding Author); Weegar, J.; Dahlbacka, J.; Kalkkinen, Nisse; Fagervik, K.; Leisola, Matti; Von Weymarn, Niklas.

In: Biotechnology and Applied Biochemistry, Vol. 44, No. 3, 2006, p. 151-158.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy

AU - Siren, N.

AU - Weegar, J.

AU - Dahlbacka, J.

AU - Kalkkinen, Nisse

AU - Fagervik, K.

AU - Leisola, Matti

AU - Von Weymarn, Niklas

PY - 2006

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N2 - In the present paper we describe the cloning and extracellular expression of the HIV‐1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low‐temperature fed‐batch strategy for decreasing end‐product degradation by proteases. The nef gene in a pPICZαA vector was integrated into the genome of three different P. pastoris strains, namely X‐33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild‐type strain (X‐33) was found to be the best choice. The decreased end‐product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44‐fold utilizing the low‐temperature strategy compared with the standard fermentation. Purification of histidine‐tagged Nef was performed in one step using a Ni2+‐nitrilotriacetate–Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix‐assisted laser‐desorption ionization–time‐of‐flight MS, reversed‐phase HPLC and N‐terminal‐sequence analysis.

AB - In the present paper we describe the cloning and extracellular expression of the HIV‐1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low‐temperature fed‐batch strategy for decreasing end‐product degradation by proteases. The nef gene in a pPICZαA vector was integrated into the genome of three different P. pastoris strains, namely X‐33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild‐type strain (X‐33) was found to be the best choice. The decreased end‐product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44‐fold utilizing the low‐temperature strategy compared with the standard fermentation. Purification of histidine‐tagged Nef was performed in one step using a Ni2+‐nitrilotriacetate–Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix‐assisted laser‐desorption ionization–time‐of‐flight MS, reversed‐phase HPLC and N‐terminal‐sequence analysis.

KW - Fed-batch

KW - HIV-1 negative factor (HIV-1 Nef)

KW - Methanol

KW - Pichia pastoris

KW - Protease

KW - recombinant proteins

U2 - 10.1042/BA20060001

DO - 10.1042/BA20060001

M3 - Article

VL - 44

SP - 151

EP - 158

JO - Biotechnology and Applied Biochemistry

JF - Biotechnology and Applied Biochemistry

SN - 0885-4513

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ER -