Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins

Kempe, Tomas; Kent, S.B.H.; Chow, Flora; Peterson, Susan; Sundquist, Wesley I.; L’Italien, James; Harbrecht, Douglas F.; Plunkett, David; DeLorbe, W.

doi:10.1016/0378-1119(85)90318-x

Cited by 48 publications

(19 citation statements)

References 13 publications

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“…The PCR fragment was digested with SfiI and ClaI, and was then inserted into pLNCX vector (Clontech, Mountain View, CA, USA) to form pLNCX-1G. The eight tandemly repeated protein G C2 domain plasmids were constructed using the Tomas Kempe’s method 50 . Briefly, each of the pLNCX-1G plasmids was digested by BamHI + XhoI and BamHI + SalI.…”

Section: Methodsmentioning

confidence: 99%

Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Hao

Chen

et al. 2017

Sci Rep

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The sensitivities of solid-phase immunoassays are limited by the quantity of detection antibodies bound to their antigens on the solid phase. Here, we developed a poly-protein G-expressing bacterium as an antibody-trapping microparticle to enhance the signals of immunoassays by increasing the accumulation of detection antibodies on the given antigen. Eight tandemly repeated fragment crystallisable (Fc) binding domains of protein G were stably expressed on the surface of Escherichia coli BL21 cells (termed BL21/8G). BL21/8G cells showed a higher avidity for trapping antibodies on their surface than monomeric protein G-expressing BL21 (BL21/1G) cells did. In the sandwich enzyme-linked immunosorbent assay (ELISA), simply mixing the detection antibody with BL21/8G provided a detection limit of 6 pg/mL for human interferon-α (IFN-α) and a limit of 30 pg/mL for polyethylene glycol (PEG)-conjugated IFN-α (Pegasys), which are better than that of the traditional ELISA (30 pg/mL for IFN-α and 100 pg/mL for Pegasys). Moreover, the sensitivity of the Western blot for low-abundance Pegasys (0.4 ng/well) was increased by 25 folds upon mixing of an anti-PEG antibody with BL21/8G cells. By simply being mixed with a detection antibody, the poly-protein G-expressing bacteria can provide a new method to sensitively detect low-abundance target molecules in solid-phase immunoassays.

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Section: Methodsmentioning

confidence: 99%

Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Hao

Chen

et al. 2017

Sci Rep

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“…The lyophilized powder of CA-Hse-COOHcontaining material was treated with concentrated TFA (20 to 50 mg/ml for 1 h at room temperature) to convert the C-terminal homoserine into the lactone form (11,16). Excess TFA was removed under a vacuum, and the residual material was solubilized in dimethyl sulfoxide (dried over molecular sieves) to a protein concentration of 1 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity

Callaway

Lai

Haselbeck

et al. 1993

Antimicrob Agents Chemother

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Cecropin A is a naturally occurring peptide with bactericidal activity against gram-negative and grampositive bacteria. Production of large quantities of bactericidal peptides that are similar in structure and activity to cecropin A has been achieved by combining recombinant DNA techniques and chemical modification. Expression of the bactericidal peptide in Escherichia coli was accomplished through the formation of a fusion protein. The 5' end of the L-ribulokinase gene was fused to a single copy of a synthetic gene encoding cecropin A. A methionine codon was engineered between the two genes, and a methionylglycine extension was introduced at the C terminus of cecropin A. Cyanogen bromide treatment of the fusion protein yielded cecropin A with a C-terminal homoserine. The recombinant cecropin A with a homoserine at the C terminus did not kill most gram-positive bacteria tested. However, recombinant cecropin A with a chemically modified C terminus has antimicrobial activity similar to that of cecropin produced by cecropia pupae.

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“…Two different recombinant methods were used to synthesize and express direct tandem repeats of I27 monomers, with identical results. The first method is a multiple-step cloning technique based on a previously described strategy (16). First, the I27 domain monomer unit was subcloned after PCR amplification of a human cardiac muscle cDNA clone (10).…”

Section: Methodsmentioning

confidence: 99%

Mechanical and chemical unfolding of a single protein: A comparison

Carrión‐Vázquez

Oberhauser

Fowler

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

985

1,196

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Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Individually folded domains are common building blocks of proteins (1, 2). The native state of proteins is the most stable, and therefore, proteins rarely unfold spontaneously. For example, the unfolding of isolated Ig and fibronectin type III domains is a rare event estimated to occur at a rate of 10, whereas refolding is typically much faster, at rates of Ϸ1 to 100 s Ϫ1 (3-6). Hence, unfolding is typically studied by using chemical denaturation, which forces the domains into various degrees of unfolding. By using protein engineering combined with a variety of spectroscopic techniques such as NMR and fluorescence, it is possible to examine the folding of protein domains after chemical denaturation (3, 7-9). These experimental approaches are widely used and give information about the folding free energy, transition state, and folding landscape.The atomic force microscope (AFM) is a simple instrument capable of causing the unfolding of a single protein by controlling its length with Å-scale resolution (10, 11). AFM techniques trigger unfolding by applying force to a single protein, which increases the rate of unfolding exponentially, thus making it easily observable without requiring chemical denaturants (10-12). However, despite these developments, single protein recordings by using AFM have remained limited, because when stretching the whole or part of a multimodular protein, it has been impossible to assign experimental observables to individual domains because of their heterogeneity. Furthermore, it is not known whether mechanical unraveling events represent true unfolding events. This has been a point that investigators have tried to address in previous papers (10, 11). However, it has been possible only to say that the unfolding and refolding rates observed and the stability measured have been ''in the range'' of the results observed for isolated domains of this kind. However, the comparison between mechanical and chemical data has remained uncertain because the range of unfolding and refolding rates of these modules varies by 2 orders of magnitude, and the stability ranges from 2 to 10 kcal͞mol (1 cal ϭ 4.18 J) (3)(4)(5)13).In this work, we use protein engineering to construct tandem repeats of a single protein module and stretch it with AFM to examine its stability and folding kinetics. Tandem repeats are necessary because the mechanical properties of a single module cannot be directly studied by using AFM techniques. A single module will extend only for a short distance and fall into...

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Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins

Cited by 48 publications

References 13 publications

Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity

Mechanical and chemical unfolding of a single protein: A comparison

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