Download lab report 1

Laboratory:
Lecture: affinity-tagged protein purification
In-Class Writing: peer review report 1 (209-210);
answer questions (page 26)
Read: Unit 2 (29-35) & JBC 266: 3811-14 (1991)
Hand In: report 1 draft
Due Next Class: lab report 1; flow chart 2
start
codon
ribosome binding site
ribosome binding site
ATG
AGA
CAT
GGA
GGC
TCC
TGA
= start codon
GGA TCG = vector codons
CAC CAT CAC CAT CAC = 6 His tag
TCC = BamHI site in vector
CAA … GTA AGC = FnbA coding sequence
GGA … TTA ATT AGC = vector codons
= stop codon
His-tagged FnbA protein:
137 amino acids
MW = 15,362
ATG
GGA
TCA
CCT
GAT
CAT
GAG
TAT
GAC
TTC
GAT
TTC
GAA
CTG
AGA
TCC
TTC
AAA
ATC
GGT
GAA
GAA
TTC
AAT
ACA
GGT
GAT
CAG
GGA
GGA
GAG
TAT
GAT
CAA
GAT
CAT
GAC
AAG
AAT
GGA
ACA
CCA
TCG
GGC
GAA
GAA
TTT
AAT
ACA
GGC
AGT
CAC
AAA
CAC
CTT
AGC
CAT
CAA
GAC
CAA
GAT
AAA
GAA
GGT
GTG
ACT
GAT
AAT
CCA
TTA
CAC
AAT
ACA
GGT
AGT
GGT
AAA
AAC
CCA
GAA
AAA
AGT
AAA
ATT
CAT
AGC
GAA
GGC
GTA
AAT
GAC
ATC
CAT
ATT
CCA
GTT
GTA
AGC
CAC
GGT
GAA
AAT
CCT
CAG
AAA
ATT
ATT
ATT
AGT
GAC
AGC
TGA
CAT
AAC
GAC
ATC
CAA
TCA
CCT
GAT
CAC
GAA
TAT
TTT
TCC
CAC
CAG
AAA
GTA
ATT
TTC
AAG
ATC
GGA
GAA
CAA
GAA
GGA
MRGSHHHHHHGSGGQNSGNQSFEE
DTEEDKPKYGQGGNIVDIDFDSVP
QIHGQNKGNQSFEEDTEKDKPKYE
HGGNIIDIDFDSVPHIHGFNKHIT
EIIEEDTNKDKPSTQFGGHNSVDF
EEDTLPKVSSGLQPSLISstop
M = start; HHHHHH = 6 His tag
RGS…GSG; SGL…LIS = vector
GQN… KVS = FnbA
Met
Gly
Ser
Pro
Asp
His
Glu
Tyr
Asp
Phe
Asp
Phe
Glu
Leu
Arg
Ser
Phe
Lys
Ile
Gly
Glu
Glu
Phe
Asn
Thr
Gly
Asp
Gln
Gly
Gly
Glu
Tyr
Asp
Gln
Asp
His
Asp
Lys
Asn
Gly
Thr
Pro
Ser
Gly
Glu
Glu
Phe
Asn
Thr
Gly
Ser
His
Lys
His
Leu
Ser
His
Gln
Asp
Gln
Asp
Lys
Glu
Gly
Val
Thr
Asp
Asn
Pro
Leu
His
Asn
Thr
Gly
Ser
Gly
Lys
Asn
Pro
Glu
Lys
Ser
Lys
Ile
His
Ser
Glu
Gly
Val
Asn
Asp
Ile
His
Ile
Pro
Val
Val
Ser
His His
Gly Asn
Glu Asn
Asn Ile
Pro Gln
Gln Ser
Lys Pro
Ile Asp
Ile His
Ile Glu
Ser Tyr
Asp Phe
Ser Ser
stop
His
Gln
Lys
Val
Ile
Phe
Lys
Ile
Gly
Glu
Gln
Glu
Gly
CL FT W E