Aloulou Ahmed, Grandval Philippe, De Caro Josiane, De Caro Alain, Carrière Frédéric
Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, UPR 9025 CNRS-Institut de Biologie Structurale et Microbiologie, Marseille, France.
Protein Expr Purif. 2006 Jun;47(2):415-21. doi: 10.1016/j.pep.2006.01.004. Epub 2006 Jan 31.
High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.
利用甲基营养型酵母毕赤酵母实现了人胰腺脂肪酶相关蛋白1（HPLRP1）的高水平组成型表达。将包含其原始前导序列的HPLRP1 cDNA亚克隆到pGAPZB载体中，并在甘油醛-3-磷酸脱氢酶（GAP）组成型启动子的控制下进一步整合到毕赤酵母X-33的基因组中。发现一种分子量为50 kDa的主要蛋白质分泌到培养基中，并用抗HPLRP1多克隆抗体鉴定为HPLRP1重组蛋白。通过特异性和定量酶联免疫吸附测定证明，培养40小时后，表达水平达到每升培养基100 - 120毫克HPLRP1。在pH 5.5条件下，将培养基中存在的HPLRP1直接批量吸附到S-Sepharose上后，单次阳离子交换色谱就足以获得高度纯化的重组HPLRP1。进行了N端测序和质谱分析，以监测成熟蛋白的产生，并确认其信号肽得到了正确加工。
