Part:BBa_I757010

b-lactamase TEM optimized

1. NgoMIV / AgeI protein fusion part
2. iGEM Team Freiburg 2007 and Jochen Hecky
3. Stabilized TEM beta-Lactamase

The sequence was based on TEM-116 (pUC lactamase) with mutations over TEM-1: V84I, A184V.

Additional mutations over TEM-116: V31A, A36L, L51I, R120G, E147G, H153R, V159T, M182T, L201P, I208M, E212K, A224V, A249V, T265M (numbering according to Ambler et al. 1991).

V31A, located in the N-terminal half of helix H1, identified in a mutant after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), increases intrinsic helix propensity.

A36L, located in the C-terminal half of helix H1, converts residue 36 to consensus, presumably increases hydrophobic contacts between helix h1 and underlying beta-sheet.

L51I, located shortly after strand S1, identified in a mutant after genetic selection for mutations compensating defects induced by circular permutation (Osuna et al., 2002) presumably improves contacts at domain interface

R120G, located at the N-terminus of helix H4, identified in mutants after genetic selection in the terminal truncation and circular permutation backgrounds, alleviates repulsive forces with helix macrodipole.

E147G, located in the N-terminal third of helix H6, identified in a mutant after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature protein (Hecky & Müller, 2005), converts residue 147 to consensus.

H153R, located at the C-terminus of helix H6, identified in mutants after genetic selection in the terminal truncation and circular permutation backgrounds, converts residue 153 to consensus.

V159T, located in a loop connecting helices H6 and H7, identified in a mutant after genetic selection for mutations compensating interface-disrupting mutations (Jochen Hecky, unpublished results).

M182T, located at the N-cap position of helix H8, identified as global suppressor mutation (Huang et al., 1997; Siederaki et al., 2001), dominant exchange in genetic selection for terminal truncation-suppressor mutations, converts residue 182 to consensus, mechanism unclear.

L201P, located at N-terminus of helix H9, identified in a mutant after genetic selection for mutations compensating interface-disrupting mutations (Jochen Hecky, unpublished results).

I208M, located in the C-terminal half of helix H9, identified in mutants after genetic selection for suppressor mutations in the terminal truncation and circular permutation backgrounds, presumably improves van der Waals contacts across the domain interface.

E212K, located at C-terminus of helix H9, identified in a mutant after genetic selection for mutations compensating defects induced by circular permutation, presumably creates favourable electrostatic interaction to D209.

A224V, resides on second crossover loop, identified in mutants after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), improves hydrophobic contacts between crossover loop, C-terminal helix H11 and the underlying beta-sheet, reinforces domain interface.

A249V, located in the C-terminal half of strand S4, chosen by visual inspection of x-ray structural model, should ideally increase packing interactions in the core.

T265M, only buried residue (< 5% accessible surface area) in the set, located at C-terminal end of strand S5, mutant identified after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), also found in clinically isolated extended spectrum lactamases, presumably acts by improving hydrophobic contacts between terminal helices and beta-sheet underneath.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 102
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 722