Amplification of the targeted genes or gene fragments were carried out by polymerase chain reaction (PCR) using the Fusion polymerase (New England Biolabs) according to the manufacturer’s manual for GC‐rich templates. Purification of the PCR products was achieved by the usage of the QIAquick PCR purification kit (Qiagen). Digestion with restriction endonucleases and ligation was carried out according to the protocol provided by the manufacturer (New England Biolabs). Plasmids were isolated as described above (4.1.2) or by usage of the QIAprep Miniprep kit (Qiagen). All constructed expression plasmids were analyzed and validated by restriction mapping and dideoxy sequencing (GATC or DNA sequencing facility Dana‐Farber Cancer Institute). Transformation of either E. coli BL21 (DE3) with plasmids containing Streptomyces genes or of E. coli BL21 Star (DE3) with plasmids containing Kutzneria genes was carried out following the manufacter’s protocol (Invitrogen).
Construction of pQTEV(asnO). The asnO gene was amplified from genomic DNA of S. coelicolor A3(2) by PCR using the oligonucleotides listed in Table 3.3. The resulting
amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pQTEV.
Construction of pQTEV(cdaPCP9). The cdaPCP9 gene fragment coding for the ninth PCP domain of the CDA peptide synthetase (cdaPSII) was amplified from genomic DNA of S.
coelicolor A3(2) by PCR using the oligonucleotides listed in Table 3.3. The resulting
amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pQTEV.
Construction of pET28a(+)(lptL). The lptL gene was amplified from genomic DNA of S. fradiae by PCR using the oligonucleotides listed in Table 3.3. The resulting amplicon was
digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pET28a(+).
Construction of pET28a(+)(lpt‐pcp3). The lpt‐pcp3 gene fragment coding for the third PCP domain of the A54145 synthetase (lptA) was amplified from genomic DNA of S.
digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pET28a(+).
Construction of pMAL‐c2X(ktzN). The ktzN gene was amplified from fosmid DNA containing the kutzneride biosynthesis cluster by PCR using the oligonucleotides listed in Table 3.3. The resulting amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pMAL‐c2X.
Construction of pQTEV(ktzO). The ktzO gene was amplified from fosmid DNA containing the kutzneride biosynthesis cluster by PCR using the oligonucleotides listed in Table 3.3. The resulting amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pQTEV.
Construction of pQTEV(ktzP). The ktzP gene was amplified from fosmid DNA containing the kutzneride biosynthesis cluster by PCR using the oligonucleotides listed in Table 3.3. The resulting amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pQTEV.
Construction of pQTEV(a*PCP3). The a*PCP3 gene fragment coding for the truncated A domain and third PCP domain of ktzH was amplified from fosmid DNA containing the kutzneride biosynthesis cluster by PCR using the oligonucleotides listed in Table 3.3. The resulting amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pQTEV.
Construction of pQTEV(PCP3). The PCP3 gene fragment coding for the third PCP domain of ktzH was amplified from fosmid DNA containing the kutzneride biosynthesis cluster by PCR using the oligonucleotides listed in Table 3.3. The resulting amplicon was digested with the corresponding enzymes (Table 3.3) and cloned into appropriate restriction sites of pQTEV. 4.1.4 Site Directed Mutagenesis The mutagenesis was carried out using the QuickChange II Kit (Stratagene) in accordance to the manufacturer’s manual. The previously described pQTEV(asnO) expression vector served as the template. A point mutation was introduced in the aspartic acid codon (GAC, bases 721‐723, D241 in the gene product) by PCR of the entire vector using the oligonucleotides listed in Table 3.3. The exchange of the 721st base guanine to adenine
resulted in an asparagine codon (AAC, bases 721‐723) and therefore the gene product is the AsnO D241N variant. The identity of the constructed mutation‐carrying plasmid pQTEV(asnOD241N) was confirmed by DNA dideoxy sequencing (GATC). 4.2 Biochemical Techniques Standard protein analysis techniques, e.g. SDS‐PAGE and gel staining, were carried out as described elsewhere.211 4.2.1 Gene Expression 4.2.1.1 Expression of pQTEV Vectors
Expressions were carried out by inoculating 500 mL LB medium supplemented with ampicillin (100 µg/mL) in a 2 L culture flask with 5 mL of an overnight culture of the corresponding production strain. Overproduction of the protein of interest was carried out on a 5 L scale. Initially, cells were grown at 34°C to an optical density of 0.6 (= 600 nm), induced with 0.1 mM isopropyl‐‐D‐thiogalactopyranoside (IPTG), and again grown at 28°C for 4 h. The cells were harvested by centrifugation (7,000 rpm, 4°C, 15 min), suspended in 15 mL buffer (50 mM HEPES, 250 mM NaCl, pH 8.0) and stored at ‐20°C.
4.2.1.2 Expression of pET28a(+) Vectors
Expressions were carried out by inoculating 500 mL LB medium supplemented with kanamycin (50 µg/mL) in a 2 L culture flask with 5 mL of an overnight culture of the corresponding production strain. Overproduction of the protein of interest was carried out on a 5 L scale. Initially, cells were grown at 37°C to an optical density of 0.5 (= 600 nm), induced with 0.1 mM IPTG, and again grown at 30°C for 4 h. The cells were harvested by centrifugation (7,000 rpm, 4°C, 15 min), suspended in 15 mL buffer (50 mM HEPES, 250 mM NaCl, pH 8.0) and stored at ‐20°C.
4.2.1.3 Expression of pMAL‐c2X Vectors
Expressions were carried out by inoculating 2 L LB medium supplemented with ampicillin (100 µg/L) and glucose (2 g/L) in a 5 L culture flask with 25 mL of an overnight culture of the corresponding production strain. Overproduction of the protein of interest was carried out on a 12 L scale. Initially, cells were incubated at 35°C, then 25°C, and finally 15°C until an optical density of 0.5 (= 600 nm) was reached. Protein expression was
induced with 0.3 mM IPTG and cultures were incubated for an additional 15 h at 15°C. The cells were harvested by centrifugation (7,000 rpm, 4°C, 15 min), suspended in 35 mL buffer (20 mM TRIS, 200 mM NaCl, 1mM EDTA, pH 7.4) and stored at ‐20°C. 4.2.2 Protein Purification 4.2.2.1 His‐tagged Proteins For purification of the His‐tagged proteins, cell suspensions were thawed and disrupted either by using an EmulsiFlex‐C5 High Pressure Homogenizer (Avestin) or a French Pressure Cell (SLM Aminco). After centrifugation (17,000 rpm, 4°C, 30 min) the supernatant was carefully removed and the recombinant protein was purified by Ni‐NTA (Qiagen) affinity chromatography using a FPLC system (Amersham Pharmacia Biotech). The Ni column was equilibrated in 50 mM HEPES, 250 mM NaCl pH 8.0 buffer, the supernatant was injected with a flow rate of 0.7 mL/min and an increasing gradient of imidazole was employed (7.5 mM to 250 mM in 30 min). Fractions containing the recombinant protein were identified by SDS‐PAGE analysis, combined, and subjected to buffer exchange into 25 mM HEPES, 50 mM NaCl, pH 7.0 using HiTrap Desalting Columns (GE Health Care). The proteins were aliquoted, flash‐frozen in liquid nitrogen and stored at ‐80°C until usage.
4.2.2.2 MBP‐tagged Proteins
For purification of the MBP‐tagged proteins, cell suspensions were thawed and disrupted by using an EmulsiFlex‐C5 High Pressure Homogenizer (Avestin). After centrifugation (17,000 rpm, 4°C, 30 min) the supernatant was carefully removed and diluted to 200 mL with column buffer (20 mM TRIS, 200 mM NaCl, 1mM EDTA, pH 7.4). The MBP‐fusion protein was purified by loading the diluted cell lysate onto an amylose gravity flow column (2.5 x 10 cm, 15 mL amylose resin, equilibrated with 8 column volumes of column buffer) with a flow rate of approximately 1 mL/min. The column was washed with 12 column volumes of column buffer. Fractional elution of the MBP‐fusion protein was carried out with column buffer containing maltose (10 mM). Fractions containing the recombinant protein were identified by SDS‐PAGE analysis, combined, and subjected to buffer exchange into 25 mM HEPES, 50 mM NaCl, 2 mM MgCl2, 10%
glycerol, pH 8.0 using HiTrap Desalting Columns (GE Health Care). The proteins were aliquoted, flash‐frozen in liquid nitrogen and stored at ‐80°C until usage.