High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA

Johnston, Sean; Lee, Jar-How; Ray, Dan S.

doi:10.1016/0378-1119(85)90121-0

Cited by 127 publications

(57 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The DNA fragment corresponding to the doubly cut vector was excised and purified from the gel. The 5Ј overhangs left by SalI digestion were filled in with Klenow and the resulting DNA was then ligated to produce what is essentially the original pING vector (24). Additionally, pUC19 empty vector was also used in the pulse-chase studies to express ␤-lactamase.…”

Section: Methodsmentioning

confidence: 99%

Altered -3 Substrate Specificity of Escherichia coli Signal Peptidase 1 Mutants as Revealed by Screening a Combinatorial Peptide Library

Ekici

Karla

Paetzel

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at ؊1 (P1) and aliphatic residues at the ؊3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the ؊1 position (Karla, Proteins destined for secretion are synthesized in a precursor form with an amino-terminal extension peptide that targets the exported protein to the Sec machinery (1) or the Tat machinery (2) in bacteria. During the export process, the signal peptide is cleaved from the precursor protein by a signal peptidase that is embedded in the plasma membrane.AIn Escherichia coli, signal peptidase (SPase I) 2 consists of a single polypeptide chain of 37 kDa (3). This enzyme spans the membrane twice with a small cytoplasmic segment (residues 29 -58) and a large carboxyl-terminal catalytic domain located in the periplasm (residues 77-323) (4 -6). Catalysis by SPase I is carried out by a Ser-Lys dyad (7-10). In the case of the E. coli SPase I, Ser-90 is the nucleophilic residue that attacks the scissile bond of the precursor substrate and lysine 145 is the general base that deprotonates the serine residue (for review, see Ref. 11). A critical serine and lysine residue is also present in SPases from other species of bacteria (12), and members of the signal peptidase I family in mitochondria (13).With the exception of the mitochondrial inner membrane peptidase I (Imp1), all type I signal peptidases carry out processing with a specificity for small aliphatic residues at the Ϫ1 (P1) and Ϫ3 (P3) positions (11). Alanine is usually the preferred amino acid residue at the Ϫ1 and Ϫ3 positions and results in the frequently observed "Ala-X-Ala" motif for signal peptide cleavage (14 -16). The residues of SPase I that comprise the substrate binding site have been identified by solving the x-ray structure of the soluble catalytic domain with a covalently attached 5S penem inhibitor (10) and a structure with a non-covalent lipohexapeptide inhibitor (17). The three-dimensional structure of SPase I with no inhibitor bound (apo-structure) revealed that there is some variation in the binding pocket volume when compared with the inhibitor-bound structures (18). The E. coli SPase residues making direct van der Waals contact with the P1 methyl group are . Those making contact with the P3 residues are Phe-84, Ile-144, Val-132, and Ile-86. The substrate binding to SPase I occurs in an extended conformation. Recently, we have made mutations of the E. coli SPase I in the S1 and S3 pockets that bind the P1 and P3 residues of the substrate to identify the residues that control the substrate specificity (19). We found that alterations of the Ile-144 and Ile-86 residues to alanine residues could alter the substrate specificity and lead to cleava...

show abstract

Section: Methodsmentioning

confidence: 99%

Altered -3 Substrate Specificity of Escherichia coli Signal Peptidase 1 Mutants as Revealed by Screening a Combinatorial Peptide Library

Ekici

Karla

Paetzel

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The second expression plasmid (pINC ssd ) (20) was a gift from Dr. Elliott Crooke at Georgetown University. This plasmid places the translation initiation sequences of bacteriophage T7 gene 10 upstream of the dnaC coding region inserted at the SmaI site of pINGK (20), a derivative constructed by insertion of the kanamycin resistance gene in a 1.2-kilobase pair PstI fragment into the PstI site of pING1 (21).…”

Section: Methodsmentioning

confidence: 99%

Essential Amino Acids of Escherichia coli DnaC Protein in an N-terminal Domain Interact with DnaB Helicase

Ludlam

McNatt

Carr

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Construction of the procoat-lep fusion (Kuhn et al, 1986) and several derivatives thereof have been described ). Pf3 -lep and its derivatives were expressed using the pING plasmid (Johnston et al, 1985), which contains the arabinose promoter and arabinose regulatory elements.…”

mentioning

confidence: 99%

The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

1995

View full text Add to dashboard Cite

The role of the membrane electrochemical potential in the translocation of acidic and basic residues across the membrane was investigated with the M13 procoat protein, which has a short periplasmic loop, and leader peptidase, which has an extended periplasmically located N‐terminal tail. For both proteins we find that the membrane potential promotes membrane transfer only when negatively charged residues are present within the translocated domain. When these residues are substituted by uncharged amino acids, the proteins insert into the membrane independently of the potential. In contrast, when a positively charged residue is present within the N‐terminal tail of leader peptidase, the potential impedes translocation of the tail domain. However, an impediment was not observed in the case of the procoat protein, where positively charged residues in the central loop are translocated even in the presence of the membrane potential. Intriguingly, several of the negatively charged procoat proteins required the SecA and SecY proteins for optimal translocation. The studies reported here provide insights into the role of the potential in membrane protein assembly and suggest that electrophoresis can play an important role in controlling membrane topology.

show abstract

High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA

Cited by 127 publications

References 24 publications

Altered -3 Substrate Specificity of Escherichia coli Signal Peptidase 1 Mutants as Revealed by Screening a Combinatorial Peptide Library

Altered -3 Substrate Specificity of Escherichia coli Signal Peptidase 1 Mutants as Revealed by Screening a Combinatorial Peptide Library

Essential Amino Acids of Escherichia coli DnaC Protein in an N-terminal Domain Interact with DnaB Helicase

The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

Contact Info

Product

Resources

About