Berkeley Structural Genomics Center

1
Berkeley Structural Genomics Center
NIGMS Protein Structure Initiative
March 7, 2002 Rosalind Kim Didier Busso Jaru
Jancarik
NIH Workshop on Protein Production for
Structural Genomics
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Challenges in Cloning and Expression     Rosalind
Kim
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Objective Goals

• Objective
• Obtain near complete structural complement of
  minimal organisms
• Mycoplasma genitalium and M. pneumoniae

• Goals
• Classify fold families
• OObtain structures for representative proteins
  from each fold family
• IInfer molecular functions for proteins with no
  known function
• AAutomate/Optimize key processes for structure
  determination

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Mycoplasma
5
Genes
6
Cellular Functions
M.p.
M.g.
ND
ND
Amino acid metabolism
Biosynthesis of cofactors
8
8
Cell envelope
54
30
20
20
Cellular processes (division, death, detox
etc.)
Chaperones
7
7
6
5
Central intermediate metabolism
Energy metabolism
39
32
Fatty acid and phopholipid metabolism
9
8
Nucleotide metabolism
19
19
Regulatory function
8
8
Replication, modification, restriction,
46
32
recombination, repair
Secretion
9
9
Transcription
13
13
Translation
99
99
Transport
44
34
Other
191
176
46
Repetitive DNA sequence derived ORFs
NA
Unique"
74
11
479
677
ND not detected NA not available
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Cloning of Mycoplasma Genes or their Homologs
Mycoplasma Gene
Homolog Gene
No UGA Codons
Mesophile/Thermophile
PCR Amplification/TOPO Cloning and Preparation of
Gene Insert
5 Expression Vectors (N-terminal Affinity Fusions)
Transformation into Expression Host
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PCR/Primer Design
NdeI Primer
CATATGNNNNNN. . . . . . . . . . . . . .
NNNNNNTAAGGATCC GTATACNNNNNN. . . . . . . . . . .
. . . NNNNNNATTCCTAGG
BamHI Primer

• Primers 24 to 45 mers (4 different suppliers)
• Three different high fidelity polymerases (Pfu,
  Deep Vent, Pfx)
• Mutation rate 15 (deletions, insertions and
  substitutions), all within
• primer region
• About 70 of mutations within the NdeI primer
  region majority
• (58) are deletions.
• Extending primer with CCC in 5 direction did
  not eliminate mutations.

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Expression Vectors Using T7 Promoter
NdeI
BamHI
1) Without fusion
pET21a
MCS
T7
2) With fusion
BamHI
NdeI
pSKB3
MCS
T7
His6
TEV
BamHI
NdeI
pDB-HisTRX
MCS
TRX
T7
His6
TEV
BamHI
NdeI
pDB-HisGST
GST
MCS
T7
His6
TEV
BamHI
NdeI
pDB-HisMBP
MBP
MCS
T7
His6
TEV
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Background Strains

BL21(DE3).STAR/pSJS1244 deficient in Lon and
OmpT proteases, RNaseE mutant stabilizes mRNA
Invitrogen pSJS1244 plasmid carries three
rare E. coli tRNA genes
(argU (AGA, AGG), ileX (AUA), lys (AAG)) BSGC
Rosetta (DE3)/pLysS.RARE deficient in Lon and
OmpT proteases, lacY1 pLysS.RARE codes for T7
lysozyme which inhibits any leaky expression
of T7 RNA polymerase and
five rare E. coli tRNA genes
(ileX (AUA), argU (AGA, AGG), leuW
(CUA),proL(CCC), glyT
(GGA)) Novagen
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Problems with Background Strain
Rosetta (DE3)/pLysS.RARE Non-Expressors
BL21(DE3) Star/pSJS1244
Soluble Insoluble Non-Expressors
19
11 (58)
5 (26)
3 (16)
58 26 84
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Lysis of Cells
MBP.MP004
MBP.MP004 (59.3 kDa)
Lysis buffer composition is important for protein
solubility.
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Salt Extraction of Insoluble Proteins
His.MP138 (45.1 kDa)
Extraction of insoluble pellet with increasing
concentration of NaCl can help solubilize the
protein.