During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

http://www.biotechlab.ch/UserFiles/File/787339_Ref_CererghinoCregg2000.pdf

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

ARP/wARP is a software suite to build macromolecular models in X-ray crystallography electron density maps. Structural genomics initiatives and the study of complex macromolecular assemblies and membrane proteins all rely on advanced methods for 3D structure determination. ARP/wARP meets these needs by providing the tools to obtain a macromolecular model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water molecules. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.

/pdf/automated-macromolecular-model-building-for-x-ray-w903jv6gcj.pdf

Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.

http://www.brasnutri.org.br/arquivos/estudos_tecnicos/Bioactive_peptides_Production_and_functionality.pdf

Bioactive peptides: Production and functionality

Vitamin B12 deficiency causes reversible megaloblastic anemia, demyelinating disease, or both. Current assays have insufficient sensitivity and specificity; methylmalonic acid levels are useful to confirm diagnosis. Parenteral or high-dose oral vitamin B12 is effective therapy.

https://www.nejm.org/doi/pdf/10.1056/NEJMcp1113996

Vitamin B12 Deficiency

Much has been published in the past 20 years on the use of measurements of arterial stiffness in animal and human research studies. This summary statement was commissioned by the American Heart Association to address issues concerning the nomenclature, methodologies, utility, limitations, and gaps in knowledge in this rapidly evolving field. The following represents an executive version of the larger online-only Data Supplement and is intended to give the reader a sense of why arterial stiffness is important, how it is measured, the situations in which it has been useful, its limitations, and questions that remain to be addressed in this field. Throughout the document, pulse-wave velocity (PWV; measured in meters per second) and variations such as carotid-femoral PWV (cfPWV; measured in meters per second) are used. PWV without modification is used in the general sense of arterial stiffness. The addition of lowercase modifiers such as “cf” is used when speaking of specific segments of the arterial circulation.

The ability to measure arterial stiffness has been present for many years, but the measurement was invasive in the early times. The improvement in technologies to enable repeated, minimal-risk, reproducible measures of this aspect of circulatory physiology led to its incorporation into longitudinal cohort studies spanning a variety of clinical populations, including those at extreme cardiovascular risk (patients on dialysis), those with comorbidities such as diabetes mellitus (DM) and hypertension, healthy elders, and general populations.

In the ≈3 decades of clinical use of PWV measures in humans, we have learned much about the importance of this parameter. PWV has proven to have independent predictive utility when evaluated in conjunction with standard risk factors for death and cardiovascular disease (CVD). However, the field of arterial stiffness investigation, which has exploded over the past 20 years, has proliferated without logistical guidance for clinical and …

Recommendations for Improving and Standardizing Vascular Research on Arterial Stiffness: A Scientific Statement From the American Heart Association

The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K(d) = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.

/pdf/functional-analyses-of-two-cellular-binding-domains-of-2tjl0328fb.pdf

Functional analyses of two cellular binding domains of bovine lactadherin.

Cobalamin (Cbl, vitamin B12) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal α6-α6 barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H2O at the upper axial side of added H2O-Cbl is displaced by a histidine residue of the α6-α6 barrel. Analysis of amino acid conservation on TC’s surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the α6-α6 barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery.

https://www.pnas.org/content/pnas/103/12/4386.full.pdf

Structural basis for mammalian vitamin B12 transport by transcobalamin

Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin.

Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin.

Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well ( r 2 = 0.87) with total cobalamin, holo-transcobalamin correlated poorly ( r 2 = 0.23) with total cobalamin or holo-haptocorrin.

Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

/pdf/direct-assay-for-cobalamin-bound-to-transcobalamin-holo-3zf10j1hmj.pdf

Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum.

BACKGROUND A novel approach to determine vitamin B12 status is to combine four blood markers: total B12 (B12), holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy). This combined indicator of B12 status is expressed as cB12=log10[(holoTC·B12)/(MMA·Hcy)]-(age factor). Here we calculate cB12 in datasets with missing biomarkers, examine the influence of folate status, and revise diagnostic cut-points. METHODS We used a database with all four markers (n=5211) plus folate measurements (n=972). A biomarker Z (assumed missing) was plotted versus X (a combination of other markers) and Y (age). Each chart was approximated by a function Ztheor, which predicted the potentially absent value(s). Statistical distributions of cB12 were aligned with physiological indicators of deficiency and used to determine cut-offs. RESULTS The predictive functions Ztheor allowed assessment of the "incomplete" indicators, 3cB12 (three markers known) and 2cB12 (two markers known). Predictions contained a systematic deviation associated with dispersion along two axes Z and X (and unaccounted by the least squares fit). Increase in tHcy at low serum folate was corrected (cB12+Δfolate) based on the function of Δfolate=log10(Hcyreal/Hcytheor) versus folate. Statistical distributions of cB12 revealed the boundaries of groups with B12 deficiency, i.e., cB12<-0.5. CONCLUSIONS We provide equations that combine two, three or four biomarkers into one diagnostic indicator, thereby rescaling unmatched data into the same coordinate system. Adjustment of this indicator is required if serum folate is <10 nmol/L and tHcy is measured. Revised cut-points and guidelines for using this approach are provided.

Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points.

We report a new method for measurement of holo-transcobalamin (holoTC), in which magnetic beads coated with vitamin B12 (cobalamins) precipitate apo-transcobalamin (apoTC) and the holoTC present in the supernatant is measured by ELISA.

Serum holoTC denotes the part of vitamin B12 accessible for the cells of the body and is considered to be a sensitive marker of vitamin B12 deficiency (1)(2)(3). Serum holoTC is not easily measured because it accounts for only approximately one-third of the circulating vitamin B12 and because the major part of TC circulates unsaturated with vitamin B12 (apoTC) (1)(2)(3). In the few published attempts to measure holoTC, TC is separated from the other vitamin B12-binding protein, haptocorrin, before quantification of vitamin B12. This allows a direct measurement of the cobalamins attached to TC (4)(5)(6) or an indirect calculation of holoTC from measurement of total plasma cobalamins and the plasma cobalamins not attached to TC (7)(8)(9). We have chosen another path.

We produced B12 beads and removed apoTC before ELISA quantification (10) of the protein moiety of holoTC. Vitamin B12 (400 mg; Fluka) was modified by acid treatment (11) to produce derivatives with a free COOH group. Magnetic beads (DB M-270 amine; DYNAL) from 10 mL of the original suspension were incubated overnight at room temperature with 10 mL of 2 mmol/L derivatives in 0.1 mol/L 2-morpholinoethane sulfonic acid, pH 5.5, in the presence of 10 mmol/L 1-ethyl-3-(dimethylaminopropyl)carbodiimide (Sigma). The beads were then washed twice with 50 mL of 1 mol/L Tris, pH 7.5, twice with 50 mL of 1 mol/L ammonium acetate, pH 4.6, and 10 times with 50 mL of 0.1 mol/L Tris–0.5 mol/L NaCl, …

/pdf/quantification-of-holo-transcobalamin-a-marker-of-vitamin-1qxvv32sb9.pdf

Sergey N. Fedosov

Papers

Functional analyses of two cellular binding domains of bovine lactadherin.

Structural basis for mammalian vitamin B12 transport by transcobalamin

Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum.

Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points.

Quantification of Holo-Transcobalamin, a Marker of Vitamin B12 Deficiency