Resultados de la cocción de placas recubiertas con esmaltes de segunda

Based on the DNA sequence of yflM from Genebank, a pair of primers were synthesised to PCR amplify yflM from samples of B. subtilis genomic DNA. The B. subtilis used in these experiments was a gift from Dr. Neil Fairweather (Imperial College of Science, Technology and Medicine, London). Bacteria were grown on L- agar plates without any antibiotics at 37°C overnight. One colony was inoculated into a 150 ml flask containing 10 ml LB and grown overnight at 37°C with vigorous shaking. The DNA extraction was carried out as described in 2.7.4. DNA was kept at 0.1 mg/ml concentration at -20°C until needed.

The sequences of the primers are:

1) 5’ primer 5’-GGGAATTCCATATGAAAGACCGTCTCGCG-3’ 2) 3’ primer 5’-GCGGGATCCTTACTCATAAGGCTTATCTTG-3’

A BamHI and an Xbal restriction enzyme site were designed into N- and C-termini of the primers to facilitate cloning in pET28a. The PCR reactions and TOPO vector cloning were performed as described in 2.7.12. Figure 3.4 shows the PCR product of

yflM amplification. yflM DNA sequence was verified according to the method described in 2.7.13. Figure 3.5 shows part o f yflM sequencing result.

Figure 3.1 Alignment of the ^yZM-encoded protein and SANOS

y f l M --- MKDRLADIKSEIDLTGSYVHTKEELEHGAKMAWRN SN 37 SANOS MLFKEAQAFIENMYKECHYETQIINKRLHDIELEI KETGTYTHTEEELIYGAKMAWRNSN 60

• • * * -k -k • -k k •k-k'k'k'k'k'k'k-k-k

y f l M RCIGRLFWNSLNVIDRRDVRTKEEVRDALFHHIET ATNNGKIRPTITIFPPEEKGEKQVE 97 SANOS RCIGRLFWDSLNVIDARDVTDEASFLSSITYHITQ ATNEGKLKPYITIYAPKD-GPK 116

★ ★ ★ ★ • • • ★ ★

y f l M IWNHQLIRYAGYESDGERIGDPASCSLTAACEELG WRGERTDFDLLPLIFRMKGDEQPVW 157

SANOS IFNNQLIRYAGYDN CGDPAEKEVTRLANHLGWKGKGTNFDVLPLIYQLP -NESVKF 171

y f l M YELPRSLVIEVPITHPDIEAFSDLELKWYGVPIIS DMKLEVGGIHYNAAPFNGWYMGTEI 217 SANOS YEYPTSLIKEVPIEHNHYPRLRKLNLKWYAVPIIS NMDLKIGGIVYPTAPFNGWYMVTEI 231

★ ★ ★ ★ ★ ★ ★ ★ • ★ ★ ★ ★ ★ • ★ ★ # # ★ ★ ★ ■*

y f l M GARNLADEKRYDKLKKVASVIGIAADYNTDLWKDQ ALVELNKAVLHSYKKQGVSIVDHHT 27 7 SANOS GVRNFIDDYRYNLLEKVADAFEFDTLKNNSFNKDR ALVELNYAVYHSFKKEGVSIVDHLT 291

•k k • k k • k • k k k • # • ★ • k k » k k k k k k k k k k » k k » k k k k k k k k

y f l M A A S Q F K R F EEQEEEAGRKLTGDWTWLIPPISPAAT HIFHRSYDNSIVKPNYFYQDK 333 SANOS A A KQFELFERNEAQQGRQVTGKWSWLAPPLSPTLT SNYHHGYDNTVKDPNFFYKKKESNA 351

★ ★ k k • k k • k • k k • k k • k • k •

y f l M PYE- 336 SANOS NQCPFHH 358

*: designates identical residues;

: : designates highly conserved residues; . : designates conserved residues.

Figure 3.2 Alignment of the j{/7M-encoded protein and human iNOS

y f l M ---

HiNOS MACPWKFLFRVKSYQGDLKEEKDINNNVEKTPGAI PSPTTQDDPKSHKHQNGFPQFLTGT 60

y f l M ---

HiNOS AQNVPESLDKLHVTPSTRPQHVRIKNWGNGEIFHD TLHHKATSDISCKSKLCMGSIMNSK 120

y f l M --- M K DRLADIKSEIDLTGSYVHTK 22 HiNOS SLTRGPRDKPTPVEELLPQAIEFINQYYGSFKEAK IEEHLARLEAVTKEIETTGTYQLTL 180

y f l M EELEHGAKMAWRNSNRCIGRLFWNSLNVIDRRDVR TKEEVRDALFHHIETATNNGKIRPT 82 HiNOS DELIFATKMAWRNAPRCIGRIQWSNLQVFDARSCS TASEMFQHICRHILYATNSGNIRSA 240

y f l M ITIFPPEEKGEKQVEIWNHQLIRYAGYES-DGERI GDPASCSLTAACEELGWRGERTDFD 141 HiNOS ITVFPQRNDGKHDFRIWNSQLIRYAGYQMPDGTIR GDPATLEFTQLCIDLGWKPRYGRFD 300

★ • ★ • • • ★ ★ ★ * * * * * * * * » * * * * * * • # * * • * * * • * *

y f l M LLPLIFRMKGDEQPVWYELPRSLVIEVPITHPDIE AFSDLELKWYGVPIISDMKLEVGGI 2 01 HiNOS VLPLVLQAHGQD-PEVFEIPPDLVLEVTMEHPKYE WFQELGLKWYALPAVANMLLEVGGL 359

• ***#•• »*•• * •*•* **•** * * * * * , * * * * * » * •••* ******

y f l M HYNAAPFNGWYMGTEIGARNLADEKRYDKLKKVAS VIGIAADYNTDLWKDQALVELNKAV 2 61 HiNOS EFPACPFNGWYMGTEIGVRDLCDTQRYNILEEVGR RMGLGTHTLASLWKDRAVTEINAAV 419

• * ************ *•* * •**• *••* •*• • • ****•*• *•* * *

y f l M LHSYKKQGVSIVDHHTAASQFKRFEEQEEEAGRKL TGDWTWLIPPISPAATHIFHRSYDN 321 HiNOS LHSFQKQNVTIMDHHTASESFMKHMQNEYRARGGC PADWIWLVPPVSGSITPVFHQEMLN 479

* * * • • * * * • * • * * * * * • * • . • * * * * * * . * * . * • * • * * . *

y f l M S I V K PNYFYQDKPYE--- 336 HiNOS YVLSPFYYYQIEPWKTHIWQDEKLRPRRREIRFTV LVKAVFFASVLMRKVMASRVRATVL 539

• • * * • * * • * • •

*: designates identical residues;

: : designates highly conserved residues; . : designates conserved residues.

Figure 3.3 NOS heme domain alignment M u r i n e iNOS Rat nNO S G u i n e a p i g iNOS H u m a n iNOS Dog iNOS C h i c k e n iNOS M o u s e nNOS Ra b b i t iNOS B u l l f r o g nNO S H u m a n eNOS Gu i n e a p i g eNOS Dog nNOS Pig iNOS Bo v i n e eNOS Sn ail NOS D r o s o p h i l a NOS M o s q u i t o NOS H o r n w o r m NOS SAN OS (S. aureus) Y F L M {B. subtilis) W R N A P R C I G R I Q W W R N A P R C I G R I Q W W R N A P R C I G R I Q W W R N A P R C I G R I Q W W R N A P R C I G R I Q W W R N A P R C I G R I Q W W R N A S R C V G R I Q W W R N A S R C V G R I Q W W R N A S R C V G R I Q W W R N A P R C V G R I Q W W R N A P R C V G R I Q W W R N A P R C V G R I Q W W R N A P R C V G R I Q W W R N A P R C V G R I Q W W R N A P G C I G R S Q W W R N S S R C I G R I Q W W R N A P R C I G R I Q W W R N A T R C I G R I Q W W R N S N R C I G R L F W W R N S N R C I G R L F W

*: designates identical residues;

: : designates highly conserved residues; . : designates conserved residues.

Figure 3.4 PCR amplifîcation oïyJlM

2016 bp

1008 bp

550 bp

Figure 3.5 j/ZAf DNA sequencing

The sequencing results oïyflM mq from last pair 727- 827 (from bottom to top). The sequence is:

GATTACAATACGGATTTATGGAAGGATCAAGCGC TAGTTGAATT GAATAAAGCTGTGCTGCACT CGTATAAAAAGCAGGGTGTCAGCA TCGTTGACCATCA

Figure 3.6 shows the yflM DNA and peptide sequences. These sequences are in 100% agreement with the sequences deposited in the NCBI database.

3.5. Expression oiyJlM in E. coli

After sequencing, yflM was cloned into the expression vector pET28a for expression in E. coli strain BL21 (DE3). Although different growth media, (for example Terrific broth, Luria-Bertani broth, 2xYT broth, and M9 minimum medium) were used for expression, no soluble protein could be detected.

Attempts were made to re-fold the insoluble pET28a-expressed yflM-Qncodtd protein using 8M urea as a dénaturant. Cells from a 100 ml induced culture of pET28a expressing yflM was pelleted by centrifugation (13,000 xg), and resuspended with 5 ml buffer B (8 M urea, 0.1 M NaH2P0 4, O.OIM Tris.HCl pH 8.0). The sample was mixed gently for 60 minutes at room temperature and the cell debris pelletted by centrifugation for 30 minutes at 4°C at 13,000 xg. 1 ml of Ni-NTA resin slurry was added to the supernatant, mixing by rotating for 60 minutes at room temperature. The mixture was loaded into an empty column, and washed with 2x4 ml aliquots of buffer C (8M urea, 0.1 M NaH2P0 4, O.OIM Tris.HCl pH6.3). The protein was eluted with 4x0.5ml buffer D (8M urea, 0. IM NaH2P0 4, O.OIM Tris.HCl pH5.9), followed by 4x0.5 ml buffer E (8M urea, 0. IM NaH2P0 4, 0.01 M Tris.HCl pH4.5).

Unfortunately, when denatured protein was renatured by decreasing the concentration of urea, the y/7M-encoded protein invariably precipitated. Although this protein was of no further use for biochemical study, denatured purified y/7M-encoded protein could be used as a protein marker in SDS-PAGE experiments to indicate the position of yflM encoded protein when SDS-PAGE was carried out

A series of additional vectors were used to attempt to express soluble 3^A/-encoded protein in E. coli. These included pET21b, pETl la, pProxHT, pTrc99a, pGEX4T-2 and pCWori. The characteristics of the different vectors are shown in 2.5.4, and the data on the primers used for yflM expression are shown in Appendix 5. Expression

Figure 3.6 Deduced j{/?M-encoded protein sequence