Practical on-line determination of biopolymer molecular weights by high-performance liquid chromatography with classical light-scattering detection

Dollinger, Gavin; Cunico, Bob; Kunitani, Michael; Johnson, D.; Jones, Robert L.

doi:10.1016/0021-9673(92)85088-b

Cited by 47 publications

(25 citation statements)

References 24 publications

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“…Furthermore, the relation holds only if the proteins are small compared to the wavelength of the light (in our case 658 nm) in order not to cause an angular dependency of the light scattering. This condition is satisfied by globular proteins with molecular masses up to several million Dalton, usually well within the range of proteins complexes studied [5,6].…”

Section: Soluble Proteinsmentioning

confidence: 74%

“…However, usually the detectors are connected in-line with a chromatography system and all three signals are measured continuously during a size exclusion chromatography (SEC) run. The method is then referred to as SEC-MALLS (Multi Angle Laser Light Scattering) [10], SEC-LALLS (Low Angle Laser Light Scattering) [7,8] or SEC-LS (Light Scattering) [5,6]. There are two reasons why direct coupling of the measurements to SEC is preferred.…”

Section: Why Couple To Size Exclusion Chromatography?mentioning

confidence: 99%

“…Here we will illustrate the use of the latter technique. Because several groups have reviewed the method of static light scattering for protein analysis in recent years [5][6][7][8][9][10][11] we will focus specifically on issues related to the analysis of membrane proteins. The relevant theory of static light scattering for membrane protein analysis will be presented, followed by an overview of practical aspects illustrated by a number of examples from our lab.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Static light scattering to characterize membrane proteins in detergent solution

Slotboom

Duurkens

Olieman

et al. 2008

Methods

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176

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Section: Soluble Proteinsmentioning

confidence: 74%

Section: Why Couple To Size Exclusion Chromatography?mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Static light scattering to characterize membrane proteins in detergent solution

Slotboom

Duurkens

Olieman

et al. 2008

Methods

166

176

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“…In addition, the high shear force induced during the ultracentrifugation may cause the dissociation of noncovalently associated HIV envelope subunits. In contrast, classical light scattering can be performed quickly and relatively easily and under very mild conditions; therefore, it is a reasonable choice for determining the molecular mass of the proteins (17,36,43). We previously used this approach to successfully characterize o-gp140-US4 and demonstrated that the purified protein was potentially in trimeric conformation (75).…”

Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Immunogenicity of a Soluble Trimeric Envelope Protein Containing a Partial Deletion of the V2 Loop Derived from SF162, an R5-Tropic Human Immunodeficiency Virus Type 1 Isolate

Srivastava

Stamatatos

Kan

et al. 2003

J Virol

146

130

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The envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the major target of neutralizing antibody responses and is likely to be a critical component of an effective vaccine against AIDS. Although monomeric HIV envelope subunit vaccines (gp120) have induced high-titer antibody responses and neutralizing antibodies against laboratory-adapted HIV-1 strains, they have failed to induce neutralizing antibodies against diverse heterologous primary HIV isolates. Most probably, the reason for this failure is that the antigenic structure(s) of these previously used immunogens does not mimic that of the functional HIV envelope, which is a trimer, and thus these immunogens do not elicit high titers of relevant functional antibodies. We recently reported that an Env glycoprotein immunogen (o-gp140SF162⌬V2) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162, when used in a DNA priming-protein boosting vaccine regimen in rhesus macaques, induced neutralizing antibodies against heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIV SF162P4 virus. Here we describe the purification of this protein to homogeneity, its characterization as trimer, and its ability to induce primary isolate-neutralizing responses in rhesus macaques. Optimal mutations in the primary and secondary protease cleavage sites of the env gene were identified that resulted in the stable secretion of a trimeric Env glycoprotein in mammalian cell cultures. We determined the molecular mass and hydrodynamic radius (R h ) using a triple detector analysis (TDA) system. The molecular mass of the oligomer was found to be 324 kDa, close to the expected M w of a HIV envelope trimer protein (330 kDa), and the hydrodynamic radius was 7.27 nm. Negative staining electron microscopy of o-gp140SF162⌬V2 showed that it is a trimer with considerable structural flexibility and supported the data obtained by TDA. The structural integrity of the purified trimeric protein was also confirmed by determinations of its ability to bind the HIV receptor, CD4, and its ability to bind a panel of well-characterized neutralizing monoclonal antibodies. No deleterious effect of V2 loop deletion was observed on the structure and conformation of the protein, and several critical neutralization epitopes were preserved and well exposed on the purified o-gp140SF162⌬V2 protein. In an intranasal priming and intramuscular boosting regimen, this protein induced high titers of functional antibodies, which neutralized the vaccine strain, i.e., SF162. These results highlight a potential role for the trimeric o-gp140SF162⌬V2 Env immunogen in a successful HIV vaccine.

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“…where A i is the number concentration of component i (Dollinger et al, 1992). The relationship is subject to the underlying assumptions of the derivation.…”

Section: Measurement Of Aggregation Rate Using Classic Light Scatterimentioning

confidence: 99%

A new kinetic scheme for lysozyme refolding and aggregation

Buswell

Middelberg

2003

Biotech & Bioengineering

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The competing first- and third-order reaction scheme for lysozyme is shown to not predict fed-batch lysozyme refolding when the model is parameterized using independent batch experiments, even when variations in chemical composition during the fed-batch experiment are accounted for. A new kinetic scheme is proposed that involves rapid partitioning between the alternative fates of refolding and aggregation, and which allows for aggregation via a sequential mechanism. The model assumes that monomeric lysozyme in different states, including native, is able to aggregate with intermediates, accounting for recent experimental evidence that native protein can be incorporated into aggregates and explaining why native protein in the refolding buffer reduces yield. Stopped-flow light-scattering measurements were used to measure the association rate for the sequential aggregation mechanism, and refolding rate constants were determined in a series of batch experiments designed to be "snapshots" of the composition during a fed-batch experiment. The new kinetic scheme gave a good a priori prediction of fed-batch refolding performance.

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Practical on-line determination of biopolymer molecular weights by high-performance liquid chromatography with classical light-scattering detection

Cited by 47 publications

References 24 publications

Static light scattering to characterize membrane proteins in detergent solution

Static light scattering to characterize membrane proteins in detergent solution

Purification, Characterization, and Immunogenicity of a Soluble Trimeric Envelope Protein Containing a Partial Deletion of the V2 Loop Derived from SF162, an R5-Tropic Human Immunodeficiency Virus Type 1 Isolate

A new kinetic scheme for lysozyme refolding and aggregation

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