scholarly journals Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26)

1995 ◽  
Vol 305 (3) ◽  
pp. 753-759 ◽  
Author(s):  
N Dilsiz ◽  
M J C Crabbe
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibody ◽  
Cytoplasmic Membrane ◽  
Amino Acid Level ◽  
Site Directed Mutagenesis ◽  
Eye Lens ◽  
Major Intrinsic Protein ◽  
Intrinsic Protein ◽  
C Terminus ◽  
Cell Plasma

The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane.

scholarly journals Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants.

1997 ◽  
Vol 44 (2) ◽  
pp. 275-283 ◽  
Author(s):  
K Bolewska ◽  
H Kozłowska ◽  
G Goch ◽  
B Mikołajek ◽  
A Bierzyński
Keyword(s):  
Escherichia Coli ◽  
Calcium Binding ◽  
Expression System ◽  
Total Yield ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Alpha Subunits ◽  
C Terminus ◽  
Gene Coding ◽  
Native Sequence

Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.

scholarly journals Topology and Function of CcmD in Cytochrome c Maturation

2008 ◽  
Vol 190 (10) ◽  
pp. 3489-3493 ◽  
Author(s):  
Cynthia L. Richard-Fogal ◽  
Elaine R. Frawley ◽  
Robert G. Kranz
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Cytochrome C Maturation ◽  
Atp Binding Cassette Transporter ◽  
C Terminus ◽  
N Terminus ◽  
Small Polypeptide ◽  
And Function

ABSTRACT The system I cytochrome c biogenesis pathway requires CcmD, a small polypeptide of 69 residues in Escherichia coli. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.

scholarly journals Mutational Analysis of the TonB1 Energy Coupler of Pseudomonas aeruginosa

2002 ◽  
Vol 184 (6) ◽  
pp. 1503-1513 ◽  
Author(s):  
Qixun Zhao ◽  
Keith Poole
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Mutational Analysis ◽  
Iron Acquisition ◽  
Energy Coupling ◽  
Site Directed Mutagenesis ◽  
Membrane Anchor ◽  
E Coli ◽  
C Terminus ◽  
Anchor Sequence

ABSTRACT Siderophore-mediated iron transport in Pseudomonas aeruginosa is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the tonB1 gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both P. aeruginosa and Escherichia coli. Appending it to the N terminus of the nonfunctional (in P. aeruginosa) Escherichia coli TonB protein (TonBEc) rendered TonBEc weakly active in P. aeruginosa and did not compromise the activity of this protein in E. coli. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in P. aeruginosa and E. coli. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonBEc function in E. coli, was shown here to be essential for TonB1 activity in E. coli but not in P. aeruginosa. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both P. aeruginosa and E. coli, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in E. coli but not in P. aeruginosa. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in P. aeruginosa. Several TonB1-TonBEc chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in P. aeruginosa, although chimeras possessing an E. coli C terminus were active in E. coli.

scholarly journals Developmental Regulation of the Direct Interaction between the Intracellular Loop of Connexin 45.6 and the C Terminus of Major Intrinsic Protein (Aquaporin-0)

2005 ◽  
Vol 280 (23) ◽  
pp. 22081-22090 ◽  
Author(s):  
Xun Sean Yu ◽  
Xinye Yin ◽  
Eileen M. Lafer ◽  
Jean X. Jiang
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Direct Interaction ◽  
Lens Development ◽  
Major Intrinsic Protein ◽  
Intracellular Loop ◽  
Intrinsic Protein ◽  
Lens Fibers ◽  
Loop Region ◽  
C Terminus

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a KD(app) of 7.5 and 10.3 μm, respectively. The KD(app) for the full-length loop region is 7.7 μm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.

Genome-wide Characterization of Major Intrinsic Protein (MIP) Gene Family in Brachypodium distachyon

2018 ◽  
Vol 13 (5) ◽  
pp. 536-552 ◽  
Author(s):  
Ankush Ashok Saddhe ◽  
Shweta ◽  
Kareem A. Mosa ◽  
Kundan Kumar ◽  
Manoj Prasad ◽  
...  
Keyword(s):  
Gene Family ◽  
Brachypodium Distachyon ◽  
Major Intrinsic Protein ◽  
Intrinsic Protein ◽  
Genome Wide
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