[Secretory expression and characterization of heat sensitive nuclease in Pichia pastoris]

Sheng Wu Gong Cheng Xue Bao. 2016 Jul 25;32(7):991-995. doi: 10.13345/j.cjb.150468.
[Article in Chinese]

Abstract

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.

核酸酶在生物工程领域有着重要的应用价值。本研究在优化北极虾核酸酶 (Shrimp nuclease,SNU) 基因序列的基础上,构建SNU 的毕赤酵母分泌表达载体SNU-pPICZα A 并转化酵母,以高拷贝整合转化子为基础,优化酶表达的条件,并对该酶的催化特性进行分析,结果显示SNU 可在毕赤酵母SMD1168H 中高效分泌表达,最佳诱导表达条件为:培养基BMMY pH 6.0,甲醇浓度为1%,诱导时间为72 h,诱导后粗酶液比活力为1.4×10⁵ U/mL。经过DEAE Sephadex 阴离子交换层析可纯化获得高纯度的目标蛋白,每升菌液可纯化15 mg目标蛋白,比活力达到6.291×10⁶ U/mg,该酶表观分子量为50 kDa,PNGaseF 酶切证实该酶存在糖基化现象。二价金属离子Ca²⁺、Mn²⁺、Co²⁺、Mg²⁺及还原剂DTT、β-ME 能显著地提高其水解活性,但Zn²⁺、Cu²⁺、SDS、高浓度NaCl 抑制该酶的活性,SNU 为Ca²⁺/Mg²⁺依赖型核酸酶。70 ℃处理10 min 可使该酶不可逆的失活。.

Keywords: Northern Shrimp Mg²⁺/Ca²⁺-independent nuclease; Pichia pastoris; catalytic characteristics; secretory expression optimization.

MeSH terms

  • Animals
  • Codon
  • Electrophoresis, Polyacrylamide Gel
  • Endonucleases / biosynthesis*
  • Glycoproteins
  • Hot Temperature
  • Penaeidae / enzymology*
  • Pichia / metabolism
  • Recombinant Proteins / biosynthesis*

Substances

  • Codon
  • Glycoproteins
  • Recombinant Proteins
  • Endonucleases