Expression of eukaryotic proteins in soluble form in Escherichia coli.

At the optimum temperature for its growth (37 degrees C), Escherichia coli tends to accumulate heterologous proteins in insoluble form. Fusion protein technology has been used to increase the solubility of overexpressed proteins in this organism, but with variable degrees of success. Fusion to a mutant form of DsbA (DsbAmut) confers higher levels of solubility to heterologous proteins in a reproducible way, even when E. coli is grown at 37 degrees C. We have shown this to be true with a diverse sample of eukaryotic proteins: IGF-I, IGFBP-3, 3C proteinase, TGF beta-2, sTGF beta-RII, BDNF, GDNF, mEGFBP, leptin, and GFP. In addition, we have investigated the effects of charge average and proline content on the solubility of DsbAmut fusions. Coexpression of a protein prolyl isomerase [cyclophilin (L-)] and modification of selected asparagine residues to aspartic acid appear to have beneficial effects on the accumulation of soluble heterologous proteins.

Leaderless polypeptides efficiently extracted from whole cells by osmotic shock.

Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited the unusual property of accumulating in an osmotically sensitive cellular compartment of Escherichia coli when their signal sequences were precisely removed by mutation. A mammalian protein, interleukin-1 (IL-1) receptor antagonist, behaved in a similar fashion in E. coli when its native signal sequence was deleted. These leaderless mutants (but not two control proteins overexpressed in the same system) were quantitatively extractable from whole cells by a variety of methods generally employed in the recovery of periplasmic proteins. A series of biochemical and genetic experiments showed that (i) leaderless DsbA (but not the wild type) was retained in a nonperiplasmic location; (ii) beta-galactosidase fusions to leaderless DsbA (but not to the wild type) exhibited efficient alpha complementation; (iii) none of the leaderless mutant proteins were substantially associated with cell membranes, even when they were overexpressed in cells; and (iv) leaderless DsbA was not transported to an osmotically sensitive compartment via a secA- or ftsZ-dependent mechanism. The observation that these proteins transit to some privileged cellular location by a previously undescribed mechanism(s)--absent their normal mode of (signal sequence-dependent) translocation--was unexpected. DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins. Despite a lack of obvious homologies, these proteins apparently have a common mechanism for intracellular localization. As this (putative) bacterial mechanism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.

Myosin light-chain expression during avian muscle development.

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.

Control of renin release in isolated rat glomeruli.

Glomeruli were isolated from rat kidneys using a passive sieving technique to study the mechanisms of basal and beta-adrenergic stimulated renin release. Glomeruli were enclosed within glass chambers and continuously superfused with Krebs media, or modified Krebs as described below, at a rate of 0.3 ml/min. The chamber effluent was collected in 10-minute fractions and measured for renin concentration (ng angiotensin I (A-1 generated) by radioimmunoassay. Basal renin was approximately 3 ng AI/ml/hr. Beta-adrenergic stimulation with isoproterenol (ISO), 178 micron M, increased renin concentration threefold (11 +/- 2 ng AI). The beta-blocker propranolol at 12 micron M halved ISO-stimulated renin, and at 120 micron M eliminated it. Doubling Krebs sodium concentration (280 mM) had no effect upon basal or ISO-stimulated renin release. Pretreating rast with DOCA and a high salt diet significantly reduced basal and abolished ISO-stimulated renin release. Increasing Krebs calcium (10 mM) did not affect basal but abolished ISO-stimulated renin release. Calcium-free Krebs had no significant effects. Increasing Krebs potassium (50 mM) increased basal renin fourfold (14 ng AI) while the absolute increase from basal due to ISO remained the same (23 ng AI). These results suggest that basal renin and ISO-stimulated renin are released via different mechanisms.

Lack of influence on collagen accumulation in granulation tissue with 'delayed' defibrinogenation. A study in the rabbit.

Systemic defibrinogenation using Arvin delays wound strength development and collagen accumulation in sponge-induced granulation tissue. Whether this effect is due to interference with the initial fibrin deposition or to any inhibiting action on fibroblast function has not been decided. In the present study the administration of Arvin is started after the initial formation of a fibrin framework. No effect on collagen accumulation in granulation tissue was demonstrated, suggesting that the action of Arvin on wound healing is limited to the first phase of wound healing and that a normal fibrin deposition is necessary for a normal healing process.

Interaction of the prostaglandin and renin-angiotensin systems in isolated rat glomeruli.

Renal glomeruli were isolated from rat kidneys using a passive mechanical sieving technique. Suspensions of 40-50 mg glomeruli were placed in glass chambers and superfused by a modified Krebs-Ringer solution. Effluent collections of 10-min fractions were measured for renin or prostaglandin (PG) E2 concentration using radioimmunoassays. The perfusate was altered to contain either the beta-adrenergic agonist isoproterenol, angiotensin II, or arachidonic acid. Isoproterenol at 1.78 or 8.1 X 10(-4) M produced a significant release of renin, but the concentration of PGE2 was unaffected. Isoproterenol-stimulated renin release was blocked by 1.2 X 10(-4) M propranolol but was unaffected by 6.3 X 10(-6) M meclofenamate. Angiotensin II at 4 or 40 X 10(-9) M altered neither renin nor PGE2. Arachidonic acid administered at 1.6 or 16.0 X 10(-5) M produced a marked increase in PG synthesis and stimulated a significant release of renin. Treatment of glomeruli with 6.3 X 10(-6) M meclofenamate attenuated PGE2 synthesis and abolished renin release, but 1.2 X 10(-4) M propranolol had no effect on PG synthesis or the coincident release of renin. These results give direct evidence of an interrelating mechanism between renal prostaglandins and renin release that is independent of external tubular or hemodynamic stimuli and show that the beta-adrenergic pathway of renin stimulation is independent of any modifying influence exerted by prostaglandins.

Cell composition of granulation tissue in defibrinogenated rabbits.

The influence of systemic defibrinogenation on cell composition and capillary formation in sponge-induced early granulation tissue was studied in rabbits. The tissue was studied histologically and the content of RNA, DNA and Hb was determined. Inflammatory cells were identified and quantified using histochemical and biochemical techniques. An accumulation of PMN-leucocytes was found after defibrinogenation whereas the amount of fibroblasts and capillaries was reduced.

Effect of defibrinogenation on wound strength and collagen formation. A study in the rabbit.

Wound healing was studied in Arvin-defibrinogenated rabbits by determinations of skin wound strength and by analysis of collagen content in sponge induced granulation tissue. Strength development was found to be delayed in skin wounds as was the accumulation of collagen in granulation tissue. These results indicate that the initial fibrin formation in a wound is of significance for repair.

Effect of defibrinogenation on collagen synthesis in granulation tissue. A study in the rabbit.

Collagen synthesis was studied in sponge-induced early granulation tissue from rabbits defibrinogenated by Arvin. Tissue slices were incubated in vitro with 14C-proline. The formation of 14C-hydroxyproline and the incorporation of 14C-proline into proteins was determined. The incorporation of proline into collagen and proteins was equally reduced after defibrinogenation. A possible explanation for this is a reduced number of active fibroblasts in granulation tissue formed during defibrinogenation.

Wound healing and formation of granulation tissue in normal and defibrinogenated rabbits. An experimental model and histological study.

The aim of this investigation was to develop an experimental model for studies on the significance of fibrin deposition for wound healing. Systemic defirbinogenation with Arvin was used to achieve abnormal fibrin deposition in subcutaneously implanted cellulose sponges. The formation of granulation tissue in the sponges was studied histologically. Arvin injections resulted in a rapid decrease in plasma fibrinogen concentration with a concomittant rise in fibrin-degradation products. No change in FXIIIa activity was seen. During defibrinogenation an abnormal fibrin deposition was found in the sponges. The fibrin strands appeared irregular and disrupted. The number of fibroblasts and collagen fibrils was reduced in granulation tissue formed during defibrinogenation. The model used seems to allow controlled studies on the significance of fibrin deposition for wound healing. Defibrinogenation was found to influence granulation tissue formation.