Koller G, Graumann K, Kramer W, Sara M, Jungbauer A
Institute of Applied Microbiology, Universität für Bodenkultur, Vienna, Austria.
J Chromatogr B Biomed Appl. 1995 Feb 3;664(1):107-18. doi: 10.1016/0378-4347(94)00468-k.
HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.
使用pKK233 - 2载体在大肠杆菌JM 105中表达来自HIV - 1毒株WMF 1.13的HIV - 1逆转录酶。细菌在净体积为14升的20升发酵罐中，使用含有细菌胰蛋白胨和酵母提取物的M9ZB培养基进行培养。通过添加异丙基 - β - D - 硫代半乳糖吡喃糖苷诱导逆转录酶（RT）表达后，监测酶浓度。通过蛋白质免疫印迹法检测可溶性和包涵体沉积的RT。通过透射电子显微镜确认包涵体的形成。研究了可溶性和不溶性RT的进一步纯化方法。在通过酶处理结合渗透压休克和离心进行细胞裂解后，通过尺寸排阻色谱法对上清液进行脱盐，并通过DEAE - Sepharose FF、AF - 肝素Toyopearl 650 M和Fractogel EMD TMAE 650（S）进一步纯化。通过用银染色的SDS - PAGE、非放射性RT测定和考马斯亮蓝蛋白质测定来监测纯化步骤的结果。沉淀物用6 M盐酸胍提取，澄清并常规复性后，以与可溶性RT相同的方式处理。该方法非常适合研究发酵条件以及纯化条件。RT以大致等量的可溶性和不溶性酶形式表达。
